Antibodies to lilrb2

ABSTRACT

Provided herein are various embodiments relating to antibodies that bind LILRB2. Anti-LILRB2 antibodies can be used in methods to treat disease, for example, cancer.

RELATED APPLICATIONS

The instant application is a division of U.S. application Ser. No.16/228,084, filed Dec. 20, 2018, which claims the benefit of andpriority to U.S. Provisional Application No. 62/610,050, filed Dec. 22,2017, the entire contents of which are incorporated by reference hereinfor all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 8, 2019 isnamed 14247-439-999_Sequence_Listing.txt and is 206,127 bytes in size.

BACKGROUND

Myeloid cells, such as dendritic cells and macrophages, can instruct theadaptive immune system to mount a response against tumor cells andpathogens by presenting peptide antigens to T cells while expressingimmunogenic cytokines and costimulatory signals, thereby promotingcytotoxic T cell activation and proliferation. Conversely, in a steadystate condition, myeloid cells maintain tolerance to endogenous proteinsby presenting self-antigens to T cells in the context of non-immunogenicsignals, such as regulatory cytokines, which can promote regulatory Tcells and suppress immunogenicity.

Cancer cells can evade the immune system by engaging signaling pathwaysassociated with immunosuppressive or immunoregulatory antigenpresentation. Such evasion events represent a major obstacle totherapeutic strategies that rely on promoting anti-tumor immunity.Therefore, there is a need for therapeutic compositions and methods thatprevent tumor-induced immunosuppression and promote immunogenicpresentation of tumor antigens by myeloid cells.

SUMMARY

The present invention features antibodies that specifically bind tohuman LILRB2. Also provided are compositions of the anti-LILRB2antibodies and methods of using the anti-LILRB2 antibodies andcompositions thereof.

In one aspect, the invention provides antibodies that specifically bindsto human LILRB2, wherein the antibody comprises the followingcomplementarity determining regions (CDRs): (a) a CDR-H1 comprising theamino acid sequence of JY(J)₂G(J)₂ (SEQ ID NO: 171); (b) a CDR-H2comprising the amino acid sequence of (J)₂W(J)₁₁KJ (SEQ ID NO: 172); (c)a CDR-H3 comprising the amino acid sequence of (J)₂I(J)₃TDYV(J)₃ (SEQ IDNO: 173); (d) a CDR-L1 comprising the amino acid sequence of (J)₈DLJ(SEQ ID NO: 174); (e) a CDR-L2 comprising the amino acid sequence of(J)₇ (SEQ ID NO: 175); and (f) a CDR-L3 comprising the amino acidsequence of (J)₃YJYPLJ (SEQ ID NO: 176); wherein each J is independentlya naturally occurring amino acid (see, e.g., Table 1, below).

In some embodiments, the CDR-H2 comprises the amino acid sequence ofSJW(J)₁₁KJ (SEQ ID NO: 177) and/or the CDR-L3 comprises the amino acidsequence of (J)₃YDYPLJ (SEQ ID NO: 178).

In some embodiments, the CDR-H1 comprises the amino acid sequence of:(i) TYAMGVS (SEQ ID NO: 15); (ii) JYAMGVS (SEQ ID NO: 179); (iii)TYJMGVS (SEQ ID NO: 180); (iv) TYAJGVS (SEQ ID NO: 181); (v) TYAMGJS(SEQ ID NO: 182); (vi) TYAMGVJ (SEQ ID NO: 183); or (vii) a variant ofany one of (ii) to (vi) comprising an additional J amino acid in placeof a recited amino acid that is represented by a J in SEQ ID NO: 171.Thus, when a variant sequence is aligned with SEQ ID NO: 171, itmaintains the non-J amino acids that are set forth in SEQ ID NO: 171.The additional J amino acid in the variant thus aligns with an aminoacid that is a J in SEQ ID NO: 171.

In some embodiments, the CDR-H2 comprises the amino acid sequence of:(i) SIWWNGNKYNNPSLKS (SEQ ID NO: 16); (ii) SJWWNGNKYNNPSLKS (SEQ ID NO:184); (iii) SIWJNGNKYNNPSLKS (SEQ ID NO: 185); (iv) SIWWJGNKYNNPSLKS(SEQ ID NO: 186); (v) SIWWNJNKYNNPSLKS (SEQ ID NO: 187); (vi)SIWWNGJKYNNPSLKS (SEQ ID NO: 188); (vii) SIWWNGNJYNNPSLKS (SEQ ID NO:189); (viii) SIWWNGNKJNNPSLKS (SEQ ID NO: 190); (ix) SIWWNGNKYJNPSLKS(SEQ ID NO: 191); (x) SIWWNGNKYNJPSLKS (SEQ ID NO: 192); (xi)SIWWNGNKYNNJSLKS (SEQ ID NO: 193); (xii) SIWWNGNKYNNPJLKS (SEQ ID NO:194); (xiii) SIWWNGNKYNNPSJKS (SEQ ID NO: 195); (xiv) SIWWNGNKYNNPSLKJ(SEQ ID NO: 196); or (xv) a variant of any one of (ii) to (xiv)comprising an additional J amino acid in place of a recited amino acidthat is represented by a J in SEQ ID NO: 172. Thus, when a variantsequence is aligned with SEQ ID NO: 172, it maintains the non-J aminoacids that are set forth in SEQ ID NO: 172. The additional J amino acidin the variant thus aligns with an amino acid that is a J in SEQ ID NO:172

In some embodiments, the CDR-H3 comprises the amino acid sequence of:(i) SRIIRFTDYVMDA (SEQ ID NO: 17); (ii) JRIIRFTDYVMDA (SEQ ID NO: 197);(iii) SJIIRFTDYVMDA (SEQ ID NO: 198); (iv) SRIJRFTDYVMDA (SEQ ID NO:199); (v) SRIIJFTDYVMDA (SEQ ID NO: 200); (vi) SRIIRJTDYVMDA (SEQ ID NO:201); (vii) SRIIRFTDYVJDA (SEQ ID NO: 202); (viii) SRIIRFTDYVMJA (SEQ IDNO: 203); (ix) SRIIRFTDYVMDJ (SEQ ID NO: 204); or (x) a variant of anyone of (ii) to (ix) comprising an additional J amino acid in place of arecited amino acid that is represented by a J in SEQ ID NO: 173. Thus,when a variant sequence is aligned with SEQ ID NO: 173, it maintains thenon-J amino acids that are set forth in SEQ ID NO: 173. The additional Jamino acid in the variant thus aligns with an amino acid that is a J inSEQ ID NO: 173.

In some embodiments, the CDR-L1 comprises the amino acid sequence of:(i) RASEDIYNDLA (SEQ ID NO: 18); (ii) JASEDIYNDLA (SEQ ID NO: 205);(iii) RJSEDIYNDLA (SEQ ID NO: 206); (iv) RAJEDIYNDLA (SEQ ID NO: 207);(v) RASJDIYNDLA (SEQ ID NO: 208); (vi) RASEJIYNDLA (SEQ ID NO: 209);(vii) RASEDJYNDLA (SEQ ID NO: 210); (viii) RASEDIJNDLA (SEQ ID NO: 211);(ix) RASEDIYJDLA (SEQ ID NO: 212); (x) RASEDIYNDLJ (SEQ ID NO: 213); or(xi) a variant of any one of (ii) to (x) comprising an additional Jamino acid in place of a recited amino acid that is represented by a Jin SEQ ID NO: 174. Thus, when a variant sequence is aligned with SEQ IDNO: 174, it maintains the non-J amino acids that are set forth in SEQ IDNO: 174. The additional J amino acid in the variant thus aligns with anamino acid that is a J in SEQ ID NO: 174.

In some embodiments, the CDR-L2 comprises the amino acid sequence of:(i) NANSLHT (SEQ ID NO: 19); (ii) JANSLHT (SEQ ID NO: 214); (iii)NJNSLHT (SEQ ID NO: 215); (iv) NAJSLHT (SEQ ID NO: 216); (v) NANJLHT(SEQ ID NO: 217); (vi) NANSJHT (SEQ ID NO: 218); (vii) NANSLJT (SEQ IDNO: 219); (viii) NANSLHJ (SEQ ID NO: 220); or (ix) a variant of any oneof (ii) to (viii) comprising an additional J amino acid in place of arecited amino acid that is represented by a J in SEQ ID NO: 175. Thus,when a variant sequence is aligned with SEQ ID NO: 175, it maintains thenon-J amino acids that are set forth in SEQ ID NO: 175. The additional Jamino acid in the variant thus aligns with an amino acid that is a J inSEQ ID NO: 175.

In some embodiments, the CDR-L3 comprises the amino acid sequence of:(i) QQYYDYPLT (SEQ ID NO: 20); (ii) JQYYDYPLT (SEQ ID NO: 221); (iii)QJYYDYPLT (SEQ ID NO: 222); (iv) QQJYDYPLT (SEQ ID NO: 223); (v)QQYYDYPLJ (SEQ ID NO: 224); or (vi) a variant of any one of (ii) to (v)comprising an additional J amino acid in place of a recited amino acidthat is represented by a J in SEQ ID NO: 176. Thus, when a variantsequence is aligned with SEQ ID NO: 176, it maintains the non-J aminoacids that are set forth in SEQ ID NO: 176. The additional J amino acidin the variant thus aligns with an amino acid that is a J in SEQ ID NO:176.

In some embodiments, the CDR-H2 comprises the amino acid sequence ofJIWWNGNKYNNPSLKS (SEQ ID NO: 225), or a variant thereof comprising anadditional J amino acid in place of a recited amino acid that isrepresented by a J in SEQ ID NO: 172, and/or the CDR-L3 comprises theamino acid sequence of QQYYJYPLT (SEQ ID NO: 226), or a variant thereofcomprising an additional J amino acid in place of a recited amino acidthat is represented by a J in SEQ ID NO: 176. Thus, when a variantsequence is aligned with SEQ ID NO: 176, it maintains the non-J aminoacids that are set forth in SEQ ID NO: 176. The additional J amino acidin the variant thus aligns with an amino acid that is a J in SEQ ID NO:176. In various embodiments of the above, one or more of the CDRscomprises said additional J amino acid. In additional embodiments, oneor more of the CDRs comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or moreadditional J amino acids. In these additional embodiments, each of theadditional J amino acids aligns with an amino acid that is a J in therespective generic formula for the CDR. The specifically recited aminoacids of a generic formula are maintained.

In some embodiments, the CDR-H3 comprises the amino acid sequence of(J)₂IJXJTDYV(J)₃ (SEQ ID NO: 227), wherein X is not arginine. In someembodiments, X is selected from the group consisting of aspartate,glutamate, and alanine. In some embodiments, the CDR-H3 comprises thesequence of: (i) JRIIXFTDYVMDA (SEQ ID NO: 228); (ii) SJIIXFTDYVMDA (SEQID NO: 229); (iii) SRIJXFTDYVMDA (SEQ ID NO: 230); (iv) SRIIXFTDYVMDA(SEQ ID NO: 231); (v) SRIIXJTDYVMDA (SEQ ID NO: 232); (vi) SRIIXFTDYVJDA(SEQ ID NO: 233); (vii) SRIIXFTDYVMJA (SEQ ID NO: 234); (viii)SRIIXFTDYVMDJ (SEQ ID NO: 235); or (ix) a variant of any one of (ii) to(iii) or (v) to (viii) comprising an additional J amino acid in place ofa recited amino acid that is represented by a J in SEQ ID NO: 173.

In some embodiments, the antibody comprises a variable heavy chain(V_(H)) region comprising an amino acid sequence that is at least 90%identical to the amino acid sequence of SEQ ID NO: 3, 13, 23, 33, 43,53, 63, 73, 83, 93, 103, or 113, and/or a variable light chain (V_(L))region comprising an amino acid sequence that is at least 90% identicalto the amino acid sequence of SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, 74,84, 94, 104, or 114. It is to be understood herein that if a sequence isrequired in an independent claim and a dependent claim permitsvariability in a larger sequence encompassing the sequence of theindependent claim, that the variability is not applicable to thesequence required in the independent claim,

In some embodiments, the antibody comprises a variable heavy chain(V_(H)) region comprising the amino acid sequence of SEQ ID NO: 3, 13,23, 33, 43, 53, 63, 73, 83, 93, 103, or 113, and/or a variable lightchain (V_(L)) region comprising the amino acid sequence of SEQ ID NO: 4,14, 24, 34, 44, 54, 64, 74, 84, 94, 104, or 114.

In some embodiments, the antibody comprises a heavy chain comprising anamino acid sequence that is at least 90% identical to the amino acidsequence of SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, or111, and/or a light chain comprising an amino acid sequence that is atleast 90% identical to the amino acid sequence of SEQ ID NO: 2, 22, 32,42, 52, 62, 72, 82, 92, 102, or 112.

In another aspect, the invention provides an antibody that specificallybinds to human LILRB2, wherein the antibody comprises the following sixcomplementarity determining regions (CDRs): (a) a CDR-H1 comprising theamino acid sequence of SEQ ID NO: 15; (b) a CDR-H2 comprising the aminoacid sequence of SEQ ID NO: 16; (c) a CDR-H3 comprising the amino acidsequence of SEQ ID NO: 17; (d) a CDR-L1 comprising the amino acidsequence of SEQ ID NO: 18; (e) a CDR-L2 comprising the amino acidsequence of SEQ ID NO: 19; and (f) a CDR-L3 comprising the amino acidsequence of SEQ ID NO: 20.

In some embodiments, the antibody comprises a variable heavy chain(V_(H)) region comprising an amino acid sequence that is at least 90%identical to the amino acid sequence of SEQ ID NO: 13 and a variablelight chain (V_(L)) region comprising an amino acid sequence that is atleast 90% identical to the amino acid sequence of SEQ ID NO: 14, whereinthe V_(H) region comprises three CDRs comprising the amino acidsequences of SEQ ID NOs: 15-17, and the V_(L) region comprises threeCDRs comprising the amino acid sequences of SEQ ID NOs: 18-20. In someembodiments, the antibody comprises a V_(H) region comprising the aminoacid sequence of SEQ ID NO: 13 and a V_(L) region comprising the aminoacid sequence of SEQ ID NO: 14. In some embodiments, the antibodycomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:11 and a light chain comprising the amino acid sequence of SEQ ID NO:12.

In some embodiments, the antibody comprises a V_(H) region comprising anamino acid sequence that is at least 90% identical to the amino acidsequence of SEQ ID NO: 53 and a V_(L) region comprising an amino acidsequence that is at least 90% identical to the amino acid sequence ofSEQ ID NO: 54, wherein the V_(H) region comprises three CDRs comprisingthe amino acid sequences of SEQ ID NOs: 15-17, and the V_(L) regioncomprises three CDRs comprising the amino acid sequences of SEQ ID NOs:18-20. In particular embodiments, the antibody comprises a V_(H) regioncomprising the amino acid sequence of SEQ ID NO: 53 and a variable lightchain V_(L) region comprising the amino acid sequence of SEQ ID NO: 54.In some embodiments, the antibody comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 51 and a light chain comprising theamino acid sequence of SEQ ID NO: 52.

In other embodiments, the antibody comprises a V_(H) region comprisingan amino acid sequence that is at least 90% identical to the amino acidsequence of SEQ ID NO: 63 and a V_(L) region comprising an amino acidsequence that is at least 90% identical to the amino acid sequence ofSEQ ID NO: 64, wherein the V_(H) region comprises three CDRs comprisingthe amino acid sequences of SEQ ID NOs: 15-17, and the V_(L) regioncomprises three CDRs comprising the amino acid sequences of SEQ ID NOs:18-20. In some embodiments, the antibody comprises a V_(H) regioncomprising the amino acid sequence of SEQ ID NO: 63 and a V_(L) regioncomprising the amino acid sequence of SEQ ID NO: 64. In someembodiments, the antibody comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 61 and a light chain comprising the aminoacid sequence of SEQ ID NO: 62.

In some embodiments, the antibody comprises a V_(H) region comprising anamino acid sequence that is at least 90% identical to the amino acidsequence of SEQ ID NO: 73 and a V_(L) region comprising an amino acidsequence that is at least 90% identical to the amino acid sequence ofSEQ ID NO: 74, wherein the V_(H) region comprises three CDRs comprisingthe amino acid sequences of SEQ ID NOs: 15-17, and the V_(L) regioncomprises three CDRs comprising the amino acid sequences of SEQ ID NOs:18-20. In particular embodiments, the antibody comprises a V_(H) regioncomprising the amino acid sequence of SEQ ID NO: 73 and a V_(L) regioncomprising the amino acid sequence of SEQ ID NO: 74. In someembodiments, the antibody comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 71 and a light chain comprising the aminoacid sequence of SEQ ID NO: 72.

In other embodiments, the antibody comprises a V_(H) region comprisingan amino acid sequence that is at least 90% identical to the amino acidsequence of SEQ ID NO: 83 and a V_(L) region comprising an amino acidsequence that is at least 90% identical to the amino acid sequence ofSEQ ID NO: 84, wherein the V_(H) region comprises three CDRs comprisingthe amino acid sequences of SEQ ID NOs: 15-17, and the V_(L) regioncomprises three CDRs comprising the amino acid sequences of SEQ ID NOs:18-20. In some embodiments, the antibody comprises a V_(H) regioncomprising the amino acid sequence of SEQ ID NO: 83 and a V_(L) regioncomprising the amino acid sequence of SEQ ID NO: 84. In particularembodiments, the antibody comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 81 and a light chain comprising the aminoacid sequence of SEQ ID NO: 82.

In other embodiments, the antibody comprises a V_(H) region comprisingan amino acid sequence that is at least 90% identical to the amino acidsequence of SEQ ID NO: 93 and a V_(L) region comprising an amino acidsequence that is at least 90% identical to the amino acid sequence ofSEQ ID NO: 94, wherein the V_(H) region comprises three CDRs comprisingthe amino acid sequences of SEQ ID NOs: 15-17, and the V_(L) regioncomprises three CDRs comprising the amino acid sequences of SEQ ID NOs:18-20. In some embodiments, the antibody comprises a V_(H) regioncomprising the amino acid sequence of SEQ ID NO: 93 and a V_(L) regioncomprising the amino acid sequence of SEQ ID NO: 94. In particularembodiments, the antibody comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 91 and a light chain comprising the aminoacid sequence of SEQ ID NO: 92.

In other embodiments, the antibody comprises a V_(H) region comprisingan amino acid sequence that is at least 90% identical to the amino acidsequence of SEQ ID NO: 103 and a V_(L) region comprising an amino acidsequence that is at least 90% identical to the amino acid sequence ofSEQ ID NO: 104, wherein the V_(H) region comprises three CDRs comprisingthe amino acid sequences of SEQ ID NOs: 15-17, and the V_(L) regioncomprises three CDRs comprising the amino acid sequences of SEQ ID NOs:18-20. In some embodiments, the antibody comprises a V_(H) regioncomprising the amino acid sequence of SEQ ID NO: 103 and a V_(L) regioncomprising the amino acid sequence of SEQ ID NO: 104. In particularembodiments, the antibody comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 101 and a light chain comprising the aminoacid sequence of SEQ ID NO: 102.

In other embodiments, the antibody comprises a V_(H) region comprisingan amino acid sequence that is at least 90% identical to the amino acidsequence of SEQ ID NO: 113 and a V_(L) region comprising an amino acidsequence that is at least 90% identical to the amino acid sequence ofSEQ ID NO: 114, wherein the V_(H) region comprises three CDRs comprisingthe amino acid sequences of SEQ ID NOs: 15-17, and the V_(L) regioncomprises three CDRs comprising the amino acid sequences of SEQ ID NOs:18-20. In some embodiments, the antibody comprises a V_(H) regioncomprising the amino acid sequence of SEQ ID NO: 113 and a V_(L) regioncomprising the amino acid sequence of SEQ ID NO: 114. In particularembodiments, the antibody comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 111 and a light chain comprising the aminoacid sequence of SEQ ID NO: 112.

In another aspect, the invention provides an antibody that specificallybinds to human LILRB2, wherein the antibody comprises the following sixCDRs: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 5;(b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 6; (c) aCDR-H3 comprising the amino acid sequence of SEQ ID NO: 7; (d) a CDR-L1comprising the amino acid sequence of SEQ ID NO: 8; (e) a CDR-L2comprising the amino acid sequence of SEQ ID NO: 9; and (f) a CDR-L3comprising the amino acid sequence of SEQ ID NO: 10.

In some embodiments, the antibody comprises a V_(H) region comprising anamino acid sequence that is at least 90% identical to the amino acidsequence of SEQ ID NO: 3 and a V_(L) region comprising an amino acidsequence that is at least 90% identical to the amino acid sequence ofSEQ ID NO: 4, wherein the V_(H) region comprises three CDRs comprisingthe amino acid sequences of SEQ ID NOs: 5-7, and the V_(L) regioncomprises three CDRs comprising the amino acid sequences of SEQ ID NOs:8-10. In some embodiments, the antibody comprises a V_(H) regioncomprising the amino acid sequence of SEQ ID NO: 3 and a V_(L) regioncomprising the amino acid sequence of SEQ ID NO: 4. In particularembodiments, the antibody comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 1 and a light chain comprising the aminoacid sequence of SEQ ID NO: 2.

In another aspect, the invention features an antibody that specificallybinds to human LILRB2, wherein the antibody comprises the following sixCDRs: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25;(b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26; (c) aCDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; (d) a CDR-L1comprising the amino acid sequence of SEQ ID NO: 28; (e) a CDR-L2comprising the amino acid sequence of SEQ ID NO: 29; and (f) a CDR-L3comprising the amino acid sequence of SEQ ID NO: 30.

In some embodiments, the antibody comprises a V_(H) region comprising anamino acid sequence that is at least 90% identical to the amino acidsequence of SEQ ID NO: 23 and a V_(L) region comprising an amino acidsequence that is at least 90% identical to the amino acid sequence ofSEQ ID NO: 24, wherein the V_(H) region comprises three CDRs comprisingthe amino acid sequences of SEQ ID NOs: 25-27, and the V_(L) regioncomprises three CDRs comprising the amino acid sequences of SEQ ID NOs:28-30. In some embodiments, the antibody comprises a V_(H) regioncomprising the amino acid sequence of SEQ ID NO: 23 and a V_(L) regioncomprising the amino acid sequence of SEQ ID NO: 24. In someembodiments, the antibody comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 21 and a light chain comprising the aminoacid sequence of SEQ ID NO: 22.

In another aspect, the invention features an antibody that specificallybinds to human LILRB2, wherein the antibody comprises the following sixCDRs: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 35;(b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 36; (c) aCDR-H3 comprising the amino acid sequence of SEQ ID NO: 37; (d) a CDR-L1comprising the amino acid sequence of SEQ ID NO: 38; (e) a CDR-L2comprising the amino acid sequence of SEQ ID NO: 39; and (f) a CDR-L3comprising the amino acid sequence of SEQ ID NO: 40.

In some embodiments, the antibody comprises a V_(H) region comprising anamino acid sequence that is at least 90% identical to the amino acidsequence of SEQ ID NO: 33 and a V_(L) region comprising an amino acidsequence that is at least 90% identical to the amino acid sequence ofSEQ ID NO: 34, wherein the V_(H) region comprises three CDRs comprisingthe amino acid sequences of SEQ ID NOs: 35-37, and the V_(L) regioncomprises three CDRs comprising the amino acid sequences of SEQ ID NOs:38-40. In some embodiments, the antibody comprises a V_(H) regioncomprising the amino acid sequence of SEQ ID NO: 33 and a V_(L) regioncomprising the amino acid sequence of SEQ ID NO: 34. In someembodiments, the antibody comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 31 and a light chain comprising the aminoacid sequence of SEQ ID NO: 32.

In another aspect, the invention features an antibody that specificallybinds to human LILRB2, wherein the antibody comprises the following sixCDRs: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 45;(b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 46; (c) aCDR-H3 comprising the amino acid sequence of SEQ ID NO: 47; (d) a CDR-L1comprising the amino acid sequence of SEQ ID NO: 48; (e) a CDR-L2comprising the amino acid sequence of SEQ ID NO: 49; and (f) a CDR-L3comprising the amino acid sequence of SEQ ID NO: 50.

In some embodiments, the antibody comprises a V_(H) region comprising anamino acid sequence that is at least 90% identical to the amino acidsequence of SEQ ID NO: 43 and a V_(L) region comprising an amino acidsequence that is at least 90% identical to the amino acid sequence ofSEQ ID NO: 44, wherein the V_(H) region comprises three CDRs comprisingthe amino acid sequences of SEQ ID NOs: 45-47, and the V_(L) regioncomprises three CDRs comprising the amino acid sequences of SEQ ID NOs:48-50. In some embodiments, the antibody comprises a V_(H) regioncomprising the amino acid sequence of SEQ ID NO: 43 and a V_(L) regioncomprising the amino acid sequence of SEQ ID NO: 44. In someembodiments, the antibody comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 41 and a light chain comprising the aminoacid sequence of SEQ ID NO: 42.

In some embodiments of any of the antibodies described herein, e.g.,those described above, the heavy chain of the antibody additionallycomprises a C-terminal lysine.

With respect to all of the aspects and embodiments summarized above,when a specific sequence is referenced, the invention also includesvariants of the sequences. In various embodiments, a variant can bespecified as including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or moresubstitutions of a recited sequence.

In another aspect, the invention features an antibody thatcross-competes for binding to human LILRB2 with an antibody of thepreceding aspects.

In another aspect, the invention features an antibody that specificallybinds to human LILRB2 and blocks the binding of HLA-G and/or HLA-A2 tohuman LILRB2. In some embodiments, the blocking is determined in a HLA-Gtetramer blocking assay using human monocyte-derived macrophages, humanmyeloid cells, and/or a cell line expressing LILRB2. In someembodiments, the assay includes the use of HLA-G conjugated beads. Insome embodiments, the antibody blocks HLA-G tetramer with an EC₅₀ ofless than 20 nM (e.g., less than 10 nM, less than 5 nM, less than 2 nM,less than 1.0 nM, less than 0.5 nM, or less than 0.2 nM). In someembodiments, the antibody blocks HLA-G tetramer with an EC₅₀ of lessthan 0.2 nM. In some embodiments, the blocking is determined in a HLA-A2tetramer blocking assay using human monocyte-derived macrophages. Insome embodiments, the antibody blocks HLA-A2 tetramer with an EC₅₀ ofless than 20 nM (e.g., less than 10 nM, less than 5 nM, less than 2 nM,less than 1.0 nM, less than 0.5 nM, or less than 0.2 nM). In someembodiments, the antibody blocks HLA-A2 tetramer with an EC₅₀ of lessthan 0.2 nM. In any of the foregoing embodiments, in which the antibodyspecifically binds to human LILRB2 and blocks the binding of HLA-Gand/or HLA-A2 to human LILRB2, the antibody can be any of the antibodiesof any other one or more aspects of the invention.

In another aspect, the invention provides an antibody that specificallybinds to human LILRB2, wherein said antibody is capable of converting anM2-like macrophage to an M1-like macrophage. In some embodiments, theconversion of an M2-like macrophage to an M1-like macrophage isindicated by an increased expression of one or more genes (e.g., one,two, three, four, five, or all six genes) selected from the groupconsisting of CXCL9, CXCL11, IRF1, TAP1, IL6R, and IL15. Additionally oralternatively, in some embodiments, the conversion of an M2-likemacrophage to an M1-like macrophage is indicated by a decreasedexpression of one or more genes (e.g., one, two, three, four, five, six,seven, eight, or nine genes) selected from the group consisting ofIL-10, CCL2, TGFBR2, CXCL13, IL21R, CD36, CR1, C1QB, and TGFBI. In someembodiments, the conversion of an M2-like macrophage to an M1-likemacrophage is detected using a tumor histoculture assay. In someembodiments, the conversion of an M2-like macrophage to an M1-likemacrophage is detected using a primary human macrophage assay usinghuman monocyte-derived macrophages. In some embodiments, the conversionof an M2-like macrophage to an M1-like macrophage is indicated by anincreased expression of one, two, or all three cytokines selected fromthe group consisting of TNFα, IL-1β, and IL-6, and/or the conversion ofan M2-like macrophage to an M1-like macrophage is indicated by decreasedexpression of one or both cytokines selected from the group consistingof IL-10 and CCL-2. In any of the foregoing embodiments, in which theantibody is capable of converting an M2-like macrophage to an M1-likemacrophage, the antibody can be any of the antibodies of any other oneor more aspects of the invention.

In some embodiments of any of the preceding aspects, the antibody bindsto LILRB2 with a dissociation constant (K_(D)) of less than 3.0 nM(e.g., less than 1.5 nM, less than 1.0 nM, or less than 750 μM.

In some embodiments of any of the preceding aspects, the antibody is amonoclonal antibody. In some embodiments of any of the precedingaspects, the antibody is a chimeric antibody, a humanized antibody, aCDR-grafted antibody, or a human antibody. In some embodiments of any ofthe preceding aspects, the antibody includes an Fc region selected fromthe group consisting of a native Fc region, a variant Fc region, and afunctional Fc region. In some embodiments of any of the precedingaspects, the antibody is a conjugate antibody or is detectably labeled.In some embodiments of any of the preceding aspects, the antibody is anIgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

In all aspects of the invention, if an optional more specific embodimentis not consistent with a more general embodiment, then it may beconsidered as excluded from the general embodiment.

In another aspect, the invention provides a nucleic acid moleculeencoding a polypeptide of the antibody of any of the preceding aspects.

In another aspect, the invention provides a host cell or vectorincluding a nucleic acid molecule encoding a polypeptide of the antibodyof any of the preceding aspects.

In another aspect, the invention features a pharmaceutical compositioncomprising the antibody of any of the preceding aspects or a nucleicacid molecule encoding the antibody of any of the preceding aspects.

In another aspect, the invention provides a method of treating a diseasein a subject in need thereof, the method including administering to thesubject a therapeutically effective amount of the antibody of any of thepreceding aspects. In some embodiments, the disease is a cancer (see,e.g., examples of cancer types listed elsewhere herein). In someembodiments, the method further includes administering the antibody incombination with a second therapeutic agent (e.g., an immunotherapy or acancer vaccine). In some embodiments, the second therapeutic agent is animmunotherapy comprising a PD-1 therapy and/or an ICOS therapy.

In another aspect, the invention provides a method of enhancing ananti-tumor immune response in a subject in need thereof, the methodincluding administering to the subject a therapeutically effectiveamount of any of the preceding aspects. In some embodiments, the methodfurther includes administering the antibody in combination with a secondtherapeutic agent (e.g., an immunotherapy or a cancer vaccine). In someembodiments, the second therapeutic agent is an immunotherapy comprisinga PD-1 therapy and/or an ICOS therapy.

In other aspects, the invention includes use of the antibodies,pharmaceutical compositions, and combinations for preventing, treating,ameliorating one or more symptoms of a disease or condition (e.g., asdescribed herein, for example, cancer) or the use of these agents forthe preparation of a medicament for these purposes.

In another aspect, the invention provides a kit including apharmaceutical composition comprising the antibody of any of thepreceding aspects or a nucleic acid molecule encoding the antibody ofany of the preceding aspects and instructions for use.

In a further aspect, the invention provides an oligonucleotidecomprising a nucleic acid fragment, wherein nucleic acid fragmentcomprises at least 16 consecutive nucleic acid residues of theoligonucleotide sequence of CGTCACCCTCAGTTGTCAG (SEQ ID NO: 143) orTCCGTGTAATCCAAGATGCTG (SEQ ID NO: 158). In some embodiments, the nucleicacid fragment comprises at least 16 consecutive nucleic acid residues ofthe oligonucleotide sequence of AGTCCCGTCACCCTCAGTTGTCAGGGGAG (SEQ IDNO: 169) or TCGTATCCGTGTAATCCAAGATGCTGATTTT (SEQ ID NO: 170).

The invention also includes a qPCR primer set comprising a forwardprimer and a reverse primer, wherein the forward primer comprises anucleic acid fragment comprising at least 16 consecutive nucleic acidresidues of the oligonucleotide sequence of SEQ ID NO: 143 or 169, andthe reverse primer comprises a nucleic acid fragment comprising at least16 consecutive nucleic acid residues of the oligonucleotide sequence ofSEQ ID NO: 158 or 170, wherein the qPCR primer set is optionallycomprised within a kit that further comprises a probe comprising atleast 16 consecutive residues of SEQ ID NO: 167.

In addition, the invention includes a method of quantifying a level ofLILRB2 expression in a biological sample, the method comprising: (a)obtaining cDNA derived from a biological sample; (b) performing qPCR onthe cDNA using an oligonucleotide of claim 87 or 88, or a qPCR primerset or kit of claim 89, to produce an amplification product, wherein theqPCR is specific for LILRB2; and (c) quantifying the amplificationproduct to determine the level of LILRB2 expression.

In another aspect, the invention provides use of an antibody asdescribed herein (e.g., above and elsewhere herein) for treating orpreventing a disease (e.g., cancer; see, e.g., below) in a subject inneed thereof by administration of a therapeutically effective amount theantibody to the subject.

In a further aspect, the invention includes use of a therapeuticallyeffective amount of an antibody as described herein (e.g., above andelsewhere herein) for enhancing an anti-tumor immune response in asubject in need thereof by administering an effective amount of theantibody to the subject.

In either of the above aspects, the use can further includeadministering the antibody in combination with a second therapeuticagent (e.g., an immunotherapy (e.g., a PD-1 therapy and/or an ICOStherapy) or a cancer vaccine).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing depicting a model of a LILRB2-expressing myeloidcell and a HLA-G-expressing tumor cell. A blocking anti-LILRB2 antibodyis shown between the LILRB2 expressed on the myeloid cell and the HLA-Gexpressed on the tumor cell.

FIGS. 2A-2L are flow cytometry plots showing downregulation ofmaturation markers on DCs by HLA-G tetramers. Expression of maturationmarkers was assessed by flow cytometry on LILRB2^(hi/pos) donor (FIGS.2A-2F) and LILRB2^(low/neg) donor (FIGS. 2G-2L) immature dendritic cells(iDCs) cultured in media alone (open histograms, dotted lines), ormatured with a cytokine cocktail in the absence (open histograms, solidlines) or presence (filled histograms) of HLA-G tetramer. Histograms aregated on live cells, as shown in FIGS. 2A and 2G. FIGS. 2B and 2H showLILRB2 expression; FIGS. 2C and 2I show CD11c expression; FIGS. 2D and2J show HLA-DR expression; FIGS. 2E and 2K show CD86 expression; andFIGS. 2F and 2L show CD80 expression.

FIGS. 3A and 3B are tables showing results of a chimeric anti-LILRB2screening. Shaded boxes indicate results which met threshold criteriafor each screening assay.

FIG. 4 is a graph showing results of a cell-based LILR familycross-reactivity screen of anti-LILRB2 chimeric mAbs. hLILRB2-specific(filled symbols) and cross-reactive (open symbols) antibodies from thescreen are shown. Antibodies detected with greater than two-fold bindingover isotype control mAb were identified as hLILR cross-reactiveantibodies (the dotted line represents this threshold).

FIG. 5 is a graph showing results of cell-based HLA-G blocking byanti-LILRB2 mAbs. HLA-G blocking anti-LILRB2 chimeric mAbs are shown inwhite bars and were identified as mAbs able to block at least 50% HLA-Gbinding to hLILRB2+ cells (dotted line). Non-blocking LILRB2 chimericmAbs are represented in black bars, and an isotype control mAb is in agray bar.

FIG. 6 is a graph showing results of cell-based HLA-A2 blocking byanti-LILRB2 mAbs. HLA-A2 blocking anti-LILRB2 chimeric mAbs are shown inwhite bars and were identified as mAbs able to block at least 50% HLA-A2binding to hLILRB2+ cells (dotted line). Non-blocking LILRB2 chimericmAbs are represented in black bars, and an isotype control mAb is in agray bar.

FIGS. 7A and 7B are graphs showing results of cell-based functionalassays of anti-LILRB2 mAbs. FIG. 7A shows TNFα production relative toisotype. FIG. 7B shows IL-10 production relative to isotype. Isotypecontrols are shown in gray/gray dashed lines.

FIGS. 8A and 8B are graphs showing correlation of ligand blocking vsM1-promoting cytokines by anti-LILRB2 mAbs. A positive correlationbetween HLA-G blocking (FIG. 8A) and HLA-A2 blocking (FIG. 8B) and TNFαproduced in response to anti-LILRB2 mAbs (black circles) was observed inprimary cell assays. Negative control mAbs are shown in gray.

FIGS. 9A-9C are graphs showing primary cell human-NHP cross-reactivityassessment of anti-LILRB2 mAbs. Antibodies were incubated with human(FIG. 9A), cyno (FIG. 9B), and rhesus (FIG. 9C) whole blood. Anti-LILRB2mAbs showed greater binding LILRB2+ populations including monocytes(circles) and neutrophils (squares) relative to lymphocytes (triangles).All anti-LILRB2 mAbs bound human monocytes and neutrophils, and a singleanti-LILRB2 mAb was found to exhibit cross-species binding to cyno andrhesus monocytes and neutrophils. Bars indicate mean of two donors perspecies.

FIGS. 10A and 10B are graphs showing on-cell binding assessment toCHO-expressed putative rhesus LILRB2 (LILRBb). Select anti-hLILRB2chimeric mAb (black) bound selectively to putative rhesus LILRBb (FIG.10A) in a dose-dependent and specific manner. This anti-hLILRB2 mAb didnot bind the closely related protein in rhesus, LILRBa (FIG. 10B).Isotype control mAb (gray) did not bind either cell line.

FIGS. 11A and 11B are tables showing results of the humanizedanti-LILRB2 characterization.

FIG. 12 is a graph showing results of a cell-based LILR familycross-reactivity screen of anti-LILRB2 humanized mAbs. hLILRB2-specific(filled symbols) antibodies from the screen are shown. NoLILR-cross-reactive antibodies were identified.

FIG. 13 is a graph showing cell-based affinity determination ofhumanized anti-hLILRB2 mAbs. All anti-hLILRB2 mAbs tested exhibiteddose-dependent specific binding to cell-expressed hLILRB2 (black), whilethe isotype control mAb did not bind hLILRB2 (gray).

FIG. 14 is a graph showing cell-based HLA-G blocking by anti-LILRB2humanized mAbs. Humanized anti-hLILRB2 mAbs (black, filled circles)block HLA-G:hLILRB2 interactions on primary human macrophages within thesub-nanomolar range. Isotype control mAb (gray) and non-blocking,chimeric anti-hLILRB2 mAb (open circles) did not disrupt the interactionbetween HLA-G and cell-expressed hLILRB2.

FIG. 15 is a graph showing cell-based HLA-A2 blocking by anti-LILRB2humanized mAbs. Humanized anti-hLILRB2 mAbs (black, filled circles)block HLA-A2:hLILRB2 interactions on primary human macrophages withinthe sub-nanomolar range. Isotype control mAb (gray) and non-blocking,chimeric anti-hLILRB2 mAb (open circles) did not disrupt the interactionbetween HLA-A2 and cell-expressed hLILRB2.

FIGS. 16A and 16B are graphs showing results of a cell-based functionalassay of anti-LILRB2 humanized mAbs. Humanized anti-hLILRB2 mAbs (black,filled circles) exhibit M1-promoting activity as measured by TNFαproduction (FIG. 16A) and suppressive-M2 activity as measured by areduction in IL-10 production (FIG. 16B) by LPS-stimulated HMDMs.Negative control mAbs including isotype control (gray) and non-ligandblocking anti-hLILRB2 chimeric mAb (open circles) did not show activityin this assay.

FIGS. 17A and 17B are graphs showing results of an on-cell bindingassessment to CHO-expressed rhesus LILRB2 (LILRBb). Select anti-hLILRB2humanized mAb (black) bound selectively to rhesus LILRBb (FIG. 17A) in adose-dependent and specific manner. This anti-hLILRB2 mAb did not bindthe closely related protein in rhesus, LILRBa (FIG. 17B). Isotypecontrol mAb (gray) did not bind either cell line.

FIG. 18 is a volcano plot showing log 2 (fold change) in gene expressionin response to treatment with respect to palivizumab control vs. −log 10(nominal p value). Each dot depicts the average normalized change ingene expression across all samples receiving the same treatment.Calculations were performed in MATLAB®, and p values were calculated byperforming using the ttest function (the null hypothesis is that theaverage normalized log 2 (fold change) in gene expression across allsamples for a particular treatment is 0).

FIG. 19 is a hierarchical clustering heatmap showing the log 2 (foldchange) in gene expression of each gene (row) in each treated sample(column) normalized to an IgG4 control for each kidney histoculturesample. Each gene in the list showed differential expression to at leasttwo treatments with a nominal p value less than 0.055 (see Table 7). Theexpression of Set 1 genes is generally downregulated in response totreatment (gray boxes) and the expression of Set 2 genes is generallyupregulated in response to treatment (black boxes). The noise thresholdis set at 0.3 based on housekeeping gene expression distribution. AEuclidean distance metric was used in the complete linkage clustering,which was performed in Spotfire.

FIG. 20 is a graph showing a response score for each donor to eachligand-blocking anti-LILRB2 antibodies.

FIGS. 21A and 21B are graphs showing monoculture signature scores foreach of three anti-LILRB2 antibodies for each non-responder, partialresponder, and full responder.

FIG. 22 is a graph showing binding of serum protein to antibodies overantibody concentration. No serum protein binding to LILRB2 antibodieswas observed.

FIGS. 23A-23D are graphs showing the results of a whole blood cytokinerelease assay. FIG. 23A shows IL-4 secretion, FIG. 23B shows IL-6secretion, FIG. 23C shows IL-8 secretion, and FIG. 23D shows TNFαsecretion. The assay was incubated for 24 hours at 37° C. Plasma wasthen isolated with cytokines and measured using a 10-cytokine MSD panel.Data are mean+/−SD of three donors.

FIGS. 24A-24D are graphs showing the results of a neutrophil activationassay. FIG. 24A shows CD11 b expression (as geometric mean fluorescenceintensity), FIG. 24B shows the percent of CD11 b high cells, FIG. 24Cshows CD62L expression (as geometric mean fluorescence intensity), andFIG. 24D shows the percent of CD62L low cells, each in response tovarious antibody concentrations. The assay was incubated for 2 hours at37° C. Changes in neutrophil activation markers (increase in CD11 b anddecrease in CD62L) were assessed by flow cytometry. Data are mean+/−SDof two donors.

FIG. 25 is a graph showing the serum concentration of anti-LILBR2antibody in cynomolgus monkeys over time, after a 30 mg/kg dose or athree mg/kg dose. Data are the average+/−SD of three individual monkeys.

FIGS. 26A and 26B are graphs showing peripheral blood neutrophilpopulations measured by complete blood count (CBC) assay in cynomolgusmonkeys pre-study and following dosing of anti-LILRB2 antibodies. Dataare presented as absolute number of cells (FIG. 26A) and as a percent oftotal (FIG. 26B).

FIGS. 27A and 27B are graphs showing growth of tumors in mice over timeafter inoculation with B16.SIY cells. FIG. 27A shows tumor growth inwild type (WT) mice, and FIG. 27B shows tumor growth in PirB knockout(Pirb^(−/−)) mice.

FIGS. 28A and 28B are graphs showing growth of tumors in mice over timeafter inoculation with LLC cells. FIG. 28A shows tumor growth in WTmice, and FIG. 28B shows tumor growth in Pirb^(−/−) mice.

FIGS. 29A and 29B are graphs showing growth of tumors in mice over timeafter inoculation with MC38 cells. FIG. 29A shows tumor growth in WTmice, and FIG. 29B shows tumor growth in Pirb^(−/−) mice.

FIG. 30 shows the sequences of the heavy chain (SEQ ID NO: 53) and lightchain (SEQ ID NO: 54) variable regions of J-19.h1.

FIG. 31 is a histogram of the IFNγ PD response scores from 173 tumorsamples from 80 tumors treated with palivizumab for 24 hours.

FIG. 32 is a Venn diagram and chart describing the PD response rates toJ-19.h1 across 3 indications: renal cell carcinoma, head and neckcancer, and lung cancer.

FIG. 33 is a series of graphs showing Keytruda signature scorescalculated for untreated samples, based on normalized gene expression(raw gene expression is normalized to housekeeping genes and negativecontrol probes, then log 2 transformed).

FIG. 34, left panel, is a table showing average IFNγ PD signature scorescalculated for 18 head and neck tumors in response to J-19.h1,pembrolizumab, or J-19.h1 combined with pembrolizumab.

DETAILED DESCRIPTION OF SOME EMBODIMENTS

In general, the present invention features antibodies against leukocyteimmunoglobulin-like receptor B2 (LILRB2), e.g., antibodies useful fortreating disease (e.g., cancer). This invention is based, in part, onthe discovery that it is possible to generate antibodies that aresimultaneously (1) specific to LILRB2 (e.g., in that they do not bindother LILRA and LILRB family members), (2) capable of blocking HLA-Gand/or HLA-A2 binding to LILRB2 on macrophages, and (3) capable ofpromoting a pro-inflammatory phenotype in contacted macrophages. Indeed,the present application discloses the identification of threeindependent families of such antibodies and discloses that antibodieswith the above properties are capable of inducing tumor-associatedmacrophages to exhibit anti-cancer properties. Based, in part, on theseproperties, as well as favorable pharmacokinetic and safety propertiesin animal models relevant to human physiology, the disclosed antibodiesare candidates for therapeutic use in humans.

Antibodies that specifically bind LILRB2 (e.g., human LILRB2) areprovided. Antibody heavy chains and light chains that are capable offorming antibodies that bind LILRB2 (e.g., human LILRB2) are alsoprovided. In addition, antibodies, heavy chains, and light chainscomprising one or more particular complementarity determining region(CDR) are provided. Also provided are antibodies that cross-compete forbinding to LILRB2 (e.g., human LILRB2) with any of the antibodiesdescribed herein. In some aspects, the present invention providesantibodies that specifically bind to LILRB2 (e.g., human LILRB2) andblocks the binding of HLA-G and/or HLA-A2 to human LILRB2. Also providedare antibodies that specifically bind to LILRB2 (e.g., human LILRB2) andare capable of converting an M2-like macrophage into an M1-likemacrophage. Polynucleotides encoding antibodies to LILRB2 (e.g., any ofthe LILRB2 antibodies provided herein) are provided. Polynucleotidesencoding antibody heavy chains or lights chains thereof are alsoprovided. Host cells containing polynucleotides disclosed herein arealso provided. Additionally, pharmaceutical compositions including anyof the antibodies or polynucleotides provided herein are provided.Methods of treatment using antibodies to LILRB2 are provided. Suchmethods include, but are not limited to, methods of treating cancer.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

All references cited herein, including patent applications, patentpublications, and Genbank Accession numbers are herein incorporated byreference, as if each individual reference were specifically andindividually indicated to be incorporated by reference in its entirety.

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 3rd. edition (2001) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. Current Protocols inMolecular Biology (F. M. Ausubel, et al. eds., (2003)); the seriesMethods in Enzymology (Academic Press, Inc.): PCR 2: A PracticalApproach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)),Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and AnimalCell Culture (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; CellBiology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press;Animal Cell Culture (R. I. Freshney), ed., 1987); Introduction to Celland Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press;Cell and Tissue Culture Laboratory Procedures (A. Doyle, J. B.Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbookof Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); GeneTransfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos,eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds.,1994); Current Protocols in Immunology (J. E. Coligan et al., eds.,1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRLPress, 1988-1989); Monoclonal Antibodies: A Practical Approach (P.Shepherd and C. Dean, eds., Oxford University Press, 2000); UsingAntibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold SpringHarbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D.Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principlesand Practice of Oncology (V. T. DeVita et al., eds., J.B. LippincottCompany, 1993); and updated versions thereof.

I. Definitions

Unless otherwise defined, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context or expressly indicated, singularterms shall include pluralities and plural terms shall include thesingular. For any conflict in definitions between various sources orreferences, the definition provided herein will control.

It is understood that embodiments of the invention described hereininclude “consisting” and/or “consisting essentially of” embodiments. Asused herein, the singular form “a”, “an,” and “the” includes pluralreferences unless indicated otherwise. Use of the term “or” herein isnot meant to imply that alternatives are mutually exclusive.

In this application, the use of “or” means “and/or” unless expresslystated or understood by one skilled in the art. In the context of amultiple dependent claim, the use of “or” refers back to more than onepreceding independent or dependent claim.

The terms “nucleic acid molecule,” “nucleic acid,” and “polynucleotide”may be used interchangeably, and refer to a polymer of nucleotides. Suchpolymers of nucleotides may contain natural and/or non-naturalnucleotides, and include, but are not limited to, DNA, RNA, and PNA.“Nucleic acid sequence” refers to the linear sequence of nucleotidesthat comprise the nucleic acid molecule or polynucleotide.

The terms “polypeptide” and “protein” are used interchangeably to referto a polymer of amino acid residues, and are not limited to a minimumlength. Such polymers of amino acid residues may contain natural ornon-natural amino acid residues, and include, but are not limited to,peptides, oligopeptides, dimers, trimers, and multimers of amino acidresidues. Both full-length proteins and fragments thereof areencompassed by the definition. The terms also include post-expressionmodifications of the polypeptide, for example, glycosylation,sialylation, acetylation, phosphorylation, and the like. Furthermore,for purposes of the present disclosure, a “polypeptide” refers to aprotein which includes modifications, such as deletions, additions, andsubstitutions (generally conservative in nature), to the nativesequence, as long as the protein maintains the desired activity. Thesemodifications may be deliberate, as through site-directed mutagenesis,or may be accidental, such as through mutations of hosts which producethe proteins or errors due to PCR amplification.

The term “specifically binds” to an antigen or epitope is a term that iswell understood in the art, and methods to determine such specificbinding are also well known in the art. A molecule is said to exhibit“specific binding” or “preferential binding” if it reacts or associatesmore frequently, more rapidly, with greater duration and/or with greateraffinity with a particular cell or substance than it does withalternative cells or substances. An antibody “specifically binds” or“preferentially binds” to a target if it binds with greater affinity,avidity, more readily, and/or with greater duration than it binds toother substances. For example, an antibody that specifically orpreferentially binds to an LILRB2 epitope is an antibody that binds thisepitope with greater affinity, avidity, more readily, and/or withgreater duration than it binds to other LILRB2 epitopes or non-LILRB2epitopes. It is also understood by reading this definition that, forexample, an antibody (or moiety or epitope) that specifically orpreferentially binds to a first target may or may not specifically orpreferentially bind to a second target. As such, “specific binding” or“preferential binding” does not necessarily require (although it caninclude) exclusive binding. Generally, but not necessarily, reference tobinding means preferential binding. “Specificity” refers to the abilityof a binding protein to selectively bind an antigen.

As used herein, “substantially pure” refers to material which is atleast 50% pure (that is, free from contaminants), e.g., at least 90%pure, at least 95% pure, at least 98% pure, or at least 99% pure.

The term “cross-competes” refers to competitive binding of one moleculewith another, e.g., by binding to all or part of the same epitope.Cross-competition can be determined using the experiments describedherein (e.g., biolayer interferometry), for example, by detecting nopositive response signal upon addition of a second antibody to a sensorafter a first antibody is bound to the signal. In particularembodiments, one LILRB2 antibody cross-competes another LILRB2 antibodyfor binding to LILRB2. Characterization of such cross-competitionbetween LILRB2 antibodies is described, e.g., in Example 3.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (forexample, bispecific (such as Bi-specific T-cell engagers) andtrispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

The term antibody includes, but is not limited to, fragments that arecapable of binding to an antigen, such as Fv, single-chain Fv (scFv),Fab, Fab′, di-scFv, sdAb (single domain antibody) and (Fab′)₂ (includinga chemically linked F(ab′)₂). Papain digestion of antibodies producestwo identical antigen-binding fragments, called “Fab” fragments, eachwith a single antigen-binding site, and a residual “Fc” fragment, whosename reflects its ability to crystallize readily. Pepsin treatmentyields an F(ab′)₂ fragment that has two antigen-combining sites and isstill capable of cross-linking antigen. The term antibody also includes,but is not limited to, chimeric antibodies, humanized antibodies, andantibodies of various species such as mouse, human, cynomolgus monkey,etc. Furthermore, for all antibody constructs provided herein, variantshaving the sequences from other organisms are also contemplated. Thus,if a human version of an antibody is disclosed, one of skill in the artwill appreciate how to transform the human sequence based antibody intoa mouse, rat, cat, dog, horse, etc. sequence. Antibody fragments alsoinclude either orientation of single chain scFvs, tandem di-scFv,diabodies, tandem tri-sdcFv, minibodies, etc. Antibody fragments alsoinclude nanobodies (sdAb, an antibody having a single, monomeric domain,such as a pair of variable domains of heavy chains, without a lightchain). An antibody fragment can be referred to as being a specificspecies in some embodiments (for example, human scFv or a mouse scFv).This denotes the sequences of at least part of the non-CDR regions,rather than the source of the construct.

The term “monoclonal antibody” refers to an antibody of a substantiallyhomogeneous population of antibodies, that is, the individual antibodiescomprising the population are identical except for possiblenaturally-occurring mutations that may be present in minor amounts.Monoclonal antibodies are highly specific, being directed against asingle antigenic site. Furthermore, in contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. Thus, a sample ofmonoclonal antibodies can bind to the same epitope on the antigen. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies may be made bythe hybridoma method first described by Kohler and Milstein, 1975,Nature 256:495, or may be made by recombinant DNA methods such asdescribed in U.S. Pat. No. 4,816,567. The monoclonal antibodies may alsobe isolated from phage libraries generated using the techniquesdescribed in McCafferty et al., 1990, Nature 348:552-554, for example.

The term “CDR” denotes a complementarity determining region as definedby the Kabat numbering scheme. Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991). Exemplary CDRs (CDR-L1,CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and95-102 of H3. CDRs can also be provided as shown in any one or more ofthe accompanying figures. With the exception of CDR1 in a variable heavychain region (V_(H)), CDRs generally comprise the amino acid residuesthat form the hypervariable loops. The various CDRs within an antibodycan be designated by their appropriate number and chain type, including,without limitation as: a) CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, andCDR-H3; b) CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and CDRH3; c) LCDR-1,LCDR-2, LCDR-3, HCDR-1, HCDR-2, and HCDR-3; or d) LCDR1, LCDR2, LCDR3,HCDR1, HCDR2, and HCDR3; etc. The term “CDR” is used herein to alsoencompass HVR or a “hypervariable region”, including hypervariableloops. Exemplary hypervariable loops occur at amino acid residues 26-32(L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3).(Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917.)

The term “heavy chain variable region” or V_(H) as used herein refers toa region comprising at least three heavy chain CDRs. In someembodiments, the heavy chain variable region includes the three CDRs andat least FR2 and FR3. In some embodiments, the heavy chain variableregion includes at least heavy chain HCDR1, framework (FR) 2, HCDR2,FR3, and HCDR3. In some embodiments, a heavy chain variable region alsocomprises at least a portion of an FR1 and/or at least a portion of anFR4.

The term “heavy chain constant region” as used herein refers to a regioncomprising at least three heavy chain constant domains, CH1, CH2, andCH3. Of course, non-function-altering deletions and alterations withinthe domains are encompassed within the scope of the term “heavy chainconstant region,” unless designated otherwise. Nonlimiting exemplaryheavy chain constant regions include γ, δ, and α. Nonlimiting exemplaryheavy chain constant regions also include ε and μ. Each heavy constantregion corresponds to an antibody isotype. For example, an antibodycomprising a γ constant region is an IgG antibody, an antibodycomprising a δ constant region is an IgD antibody, and an antibodycomprising an α constant region is an IgA antibody. Further, an antibodycomprising a μ constant region is an IgM antibody, and an antibodycomprising an c constant region is an IgE antibody. Certain isotypes canbe further subdivided into subclasses. For example, IgG antibodiesinclude, but are not limited to, IgG1 (comprising a γ₁ constant region),IgG2 (comprising a γ₂ constant region), IgG3 (comprising a γ₃ constantregion), and IgG4 (comprising a γ₄ constant region) antibodies; IgAantibodies include, but are not limited to, IgA1 (comprising an α₁constant region) and IgA2 (comprising an α₂ constant region) antibodies;and IgM antibodies include, but are not limited to, IgM1 and IgM2.

The term “heavy chain” as used herein refers to a polypeptide comprisingat least a heavy chain variable region, with or without a leadersequence. In some embodiments, a heavy chain comprises at least aportion of a heavy chain constant region. The term “full-length heavychain” as used herein refers to a polypeptide comprising a heavy chainvariable region and a heavy chain constant region, with or without aleader sequence.

The term “light chain variable region” of V_(L) as used herein refers toa region comprising at least three light chain CDRs. In someembodiments, the light chain variable region includes the three CDRs andat least FR2 and FR3. In some embodiments, the light chain variableregion includes at least light chain LCDR1, framework (FR) 2, LCDR2,FR3, and LCDR3. For example, a light chain variable region may compriselight chain CDR1, framework (FR) 2, CDR2, FR3, and CDR3. In someembodiments, a light chain variable region also comprises at least aportion of an FR1 and/or at least a portion of an FR4.

The term “light chain constant region” as used herein refers to a regioncomprising a light chain constant domain, C_(L). Nonlimiting exemplarylight chain constant regions include A and K. Of course,non-function-altering deletions and alterations within the domains areencompassed within the scope of the term “light chain constant region,”unless designated otherwise.

The term “light chain” as used herein refers to a polypeptide comprisingat least a light chain variable region, with or without a leadersequence. In some embodiments, a light chain comprises at least aportion of a light chain constant region. The term “full-length lightchain” as used herein refers to a polypeptide comprising a light chainvariable region and a light chain constant region, with or without aleader sequence.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain(V_(L)) framework or a heavy chain variable domain (V_(H)) frameworkderived from a human immunoglobulin framework or a human consensusframework, as defined below. An acceptor human framework derived from ahuman immunoglobulin framework or a human consensus framework cancomprise the same amino acid sequence thereof, or it can contain aminoacid sequence changes. In some embodiments, the number of amino acidchanges are 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer,5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer. In some embodiments,the V_(L) acceptor human framework is identical in sequence to the V_(L)human immunoglobulin framework sequence or human consensus frameworksequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (for example,an antibody) and its binding partner (for example, an antigen). Theaffinity of a molecule X for its partner Y can generally be representedby the equilibrium dissociation constant (K_(D)). Affinity can bemeasured by common methods known in the art (such as, for example, ELISAK_(D), KinExA, bio-layer interferometry (BLI), and/or surface plasmonresonance devices (such as a BIACORE® device), including those describedherein).

The term “K_(D)”, as used herein, refers to the equilibrium dissociationconstant of an antibody-antigen interaction.

In some embodiments, the “K_(D)” of the antibody is measured by usingsurface plasmon resonance assays using a BIACORE®-2000 or aBIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. withimmobilized antigen CM5 chips at −10 response units (RU). Briefly,carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) areactivated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, to 5 μg/ml (˜0.2 μM), before injection at a flow rate of 5μL/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, serialdilutions of polypeptide, for example, full length antibody, areinjected in PBS with 0.05% TWEEN-20™ surfactant (PBST) at 25° C. at aflow rate of approximately 25 μL/min. Association rates (k_(on)) anddissociation rates (k_(off)) are calculated using a simple one-to-oneLangmuir binding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (K_(D)) is calculated as the ratiok_(off)/k_(on). See, for example, Chen et al., 1999, J. Mol. Biol.293:865-881. If the on-rate exceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmonresonance assay above, then the on-rate can be determined by using afluorescent quenching technique that measures the increase or decreasein fluorescence emission intensity (excitation=295 nm; emission=340 nm,16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody in PBS, pH7.2, in the presence of increasing concentrations of antigen as measuredin a spectrometer, such as a stop-flow equipped spectrophometer (AvivInstruments) or a 8000-series SLM-AMINCO™ spectrophotometer(ThermoSpectronic) with a stirred cuvette.

In some embodiments, the difference between said two values (forexample, K_(D) values) is substantially the same, for example, less thanabout 50%, less than about 40%, less than about 30%, less than about20%, and/or less than about 10% as a function of thereference/comparator value.

In some embodiments, the difference between said two values (forexample, K_(D) values) is substantially different, for example, greaterthan about 10%, greater than about 20%, greater than about 30%, greaterthan about 40%, and/or greater than about 50% as a function of the valuefor the reference/comparator molecule.

“Surface plasmon resonance” denotes an optical phenomenon that allowsfor the analysis of real-time biospecific interactions by detection ofalterations in protein concentrations within a biosensor matrix, forexample using the BIAcore™ system (BIAcore International AB, a GEHealthcare company, Uppsala, Sweden and Piscataway, N.J.). For furtherdescriptions, see Jonsson et al., 1993, Ann. Biol. Clin. 51:19-26.

“Biolayer interferometry” refers to an optical analytical technique thatanalyzes the interference pattern of light reflected from a layer ofimmobilized protein on a biosensor tip and an internal reference layer.Changes in the number of molecules bound to the biosensor tip causeshifts in the interference pattern that can be measured in real-time. Anonlimiting exemplary device for biolayer interferometry is FORTEBIO®OCTET® RED96 system (Pall Corporation). See, e.g., Abdiche et al., 2008,Anal. Biochem. 377: 209-277.

The term “k_(on)”, as used herein, refers to the rate constant forassociation of an antibody to an antigen. Specifically, the rateconstants (k_(on) and k_(off)) and equilibrium dissociation constant(K_(D)) are measured using IgGs (bivalent) with monovalent antigen(e.g., LILRB2 antigen). “K_(on)”, “k_(on)”, “association rate constant”,or “ka”, are used interchangeably herein. The value indicates thebinding rate of a binding protein to its target antigen or the rate ofcomplex formation between an antibody and antigen, shown by theequation:

Antibody(“Ab”)+Antigen(“Ag”)→Ab-Ag.

The term “k_(off)”, as used herein, refers to the rate constant fordissociation of an antibody from the antibody/antigen complex. k_(off)is also denoted as “K_(off)” or the “dissociation rate constant”. Thisvalue indicates the dissociation rate of an antibody from its targetantigen or separation of Ab-Ag complex over time into free antibody andantigen as shown by the equation:

Ab+Ag

The term “biological activity” refers to any one or more biologicalproperties of a molecule (whether present naturally as found in vivo, orprovided or enabled by recombinant means). Biological propertiesinclude, but are not limited to, binding a receptor, inducing cellproliferation, inhibiting cell growth, inducing maturation or activation(e.g., myeloid cell maturation or activation), inhibiting maturation oractivation (e.g., myeloid cell maturation or activation), inducingcytokine expression or secretion (e.g., inflammatory cytokines orimmunosuppressive cytokines), inducing apoptosis, and enzymaticactivity. In some embodiments, biological activity of an LILRB2 proteinincludes, for example, conversion of M2-like macrophages to M1-likemacrophages.

An “M2-like macrophage,” as used herein, refers to a macrophagecharacterized by one or more immunosuppressive characteristics, relativeto a reference. Immunosuppressive characteristics include decreasedmaturation marker or activation marker expression (e.g., decreasedexpression of one or more costimulatory markers (e.g., CD80 or CD86),decreased antigen presentation (e.g., by HLA expression), decreasedexpression of inflammatory cytokines (e.g., TNFα, IL-6, or IL-1β), andincreased regulatory or suppressive marker expression (e.g., increasedIL-10 or CCL-2 expression or secretion). Immunosuppressivecharacteristics may additionally or alternatively be characterized bydecrease in immunogenic or inflammatory gene expression, or increase inimmunosuppressive or immunoregulatory gene expression, according tomethods known in the art. Immunosuppressive characteristics mayadditionally or alternatively be characterized by one or more functionalqualities, such as the ability to inhibit activation and/or expansion ofother immune cells. Assays suitable for identifying a macrophage as anM2-like macrophage are known in the art and described herein. Forexample, a primary human macrophage assay can be used to determinewhether a macrophage is an M2-like macrophage or an M1-like macrophage.In some instances, an M2-like macrophage is a tumor-associatedmacrophage. In the context of determining whether a macrophage is anM2-like macrophage, a reference can be provided by a control macrophageof the same or different origin (e.g., an untreated control or anLPS-treated control). In embodiments in which a candidate macrophage isa tumor-associated macrophage, a control may be a non-tumor-associatedmacrophage (e.g., from a healthy donor). Alternatively, a reference canbe a predetermined threshold, e.g., a parameter derived from anart-known immunosuppressive threshold.

An “M1-like macrophage,” as used herein, refers to a macrophagecharacterized by one or more immunogenic (e.g., immunostimulatory oractivatory) characteristics, relative to a reference. Immunogeniccharacteristics include increased maturation marker or activation markerexpression (e.g., increased expression of one or more costimulatorymarkers (e.g., CD80 or CD86), increased antigen presentation (e.g., byHLA expression), increased expression of activating cytokines (e.g.,TNFα, IL-6, or IL-1β), decreased regulatory or suppressive markerexpression (e.g., decreased IL-10 or CCL-2 expression or secretion).Immunogenic characteristics may additionally or alternatively becharacterized by increase in immunogenic or inflammatory geneexpression, or decrease in immunosuppressive or immunoregulatory geneexpression, according to methods known in the art. Immunogeniccharacteristics may additionally or alternatively be characterized byone or more functional qualities, such as the ability to activate and/orexpand other immune cells. Assays suitable for identifying a macrophageas an M1-like macrophage are known in the art and described herein. Forexample, a primary human macrophage assay can be used to determinewhether a macrophage is an M2-like macrophage or an M1-like macrophage.In some instances, an M1-like macrophage is a tumor-associatedmacrophage (e.g., a tumor-associated macrophage that has been exposed toan antibody to LILRB2). In the context of determining whether amacrophage is an M1-like macrophage, a reference can be provided by acontrol macrophage of the same or different origin (e.g., an untreatedcontrol or an immunosuppressed control). In embodiments in which acandidate macrophage is a tumor-associated macrophage, a control may bea non-tumor-associated macrophage (e.g., from a healthy donor).Alternatively, a reference can be a predetermined threshold, e.g., aparameter derived from an art-known immunogenic threshold.

“Conversion of an M2-like macrophage to an M1-like macrophage” can beidentified upon detection of an increase in any one or morecharacteristics of an M1-like macrophage, a decrease in any one or morecharacteristics of an M2-like macrophage, or any combination thereof.

As used herein, a “human monocyte-derived macrophage,” a “humanmonocyte-differentiated macrophage,” or an “HMDM” refers to a macrophagethat has been derived from a primary human monocyte. In someembodiments, the primary human macrophage is derived from monocytes fromwhole blood (e.g., from a PBMC population). In some embodiments, primaryhuman monocytes are incubated in the presence of M-CSF for seven days. Ahuman monocyte-derived macrophage can be obtained using the methodsdescribed in Example 6.

As used herein, the term “tetramer blocking assay” refers to an assayincluding the following steps:

-   -   (1) plate 1×10⁵ macrophages (e.g., human monocyte differentiated        macrophages (HMDMs)) in a well of a 96-well round-bottom tissue        culture plate;    -   (2) add 50 μL test antibody (e.g., LILRB2 antibody or isotype        control) in buffer (e.g., FACS buffer (1×DPBS containing 2%        HI-FBS (Sigma)+0.05% Sodium Azide));    -   (3) incubate 30 minutes at 4° C.;    -   (4) wash cells in buffer (e.g., FACS buffer) and resuspend in 50        μL buffer (e.g., FACS buffer) containing 1 μg/mL tetramer (e.g.,        fluorochrome-labeled tetramer, e.g., HLA-G or HLA-A2 tetramer);    -   (5) incubate protected from light for 30-60 minutes at 4° C.;    -   (6) wash cells in buffer (e.g., FACS buffer); and    -   (7) quantify tetramer binding (e.g., using flow cytometry).

A “chimeric antibody” as used herein refers to an antibody in which aportion of the heavy and/or light chain is derived from a particularsource or species, while at least a part of the remainder of the heavyand/or light chain is derived from a different source or species. Insome embodiments, a chimeric antibody refers to an antibody comprisingat least one variable region from a first species (such as mouse, rat,cynomolgus monkey, etc.) and at least one constant region from a secondspecies (such as human, cynomolgus monkey, etc.). In some embodiments, achimeric antibody comprises at least one mouse variable region and atleast one human constant region. In some embodiments, a chimericantibody comprises at least one cynomolgus variable region and at leastone human constant region. In some embodiments, all of the variableregions of a chimeric antibody are from a first species and all of theconstant regions of the chimeric antibody are from a second species. Thechimeric construct can also be a functional fragment, as noted above.

A “humanized antibody” as used herein refers to an antibody in which atleast one amino acid in a framework region of a non-human variableregion has been replaced with the corresponding amino acid from a humanvariable region. In some embodiments, a humanized antibody comprises atleast one human constant region or fragment thereof. In someembodiments, a humanized antibody is an antibody fragment, such as Fab,an scFv, a (Fab′)₂, etc. The term humanized also denotes forms ofnon-human (for example, murine) antibodies that are chimericimmunoglobulins, immunoglobulin chains, or fragments thereof (such asFv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) that contain minimal sequence of non-human immunoglobulin.Humanized antibodies can include human immunoglobulins (recipientantibody) in which residues from a complementary determining region(CDR) of the recipient are substituted by residues from a CDR of anon-human species (donor antibody) such as mouse, rat, or rabbit havingthe desired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, the humanized antibodycan comprise residues that are found neither in the recipient antibodynor in the imported CDR or framework sequences, but are included tofurther refine and optimize antibody performance. In general, thehumanized antibody can comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. In some embodiments, the humanizedantibody can also comprise at least a portion of an immunoglobulinconstant region or domain (Fc), typically that of a humanimmunoglobulin. Other forms of humanized antibodies have one or moreCDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and/or CDR H3) which arealtered with respect to the original antibody, which are also termed oneor more CDRs “derived from” one or more CDRs from the original antibody.As will be appreciated, a humanized sequence can be identified by itsprimary sequence and does not necessarily denote the process by whichthe antibody was created.

An “CDR-grafted antibody” as used herein refers to a humanized antibodyin which one or more complementarity determining regions (CDRs) of afirst (non-human) species have been grafted onto the framework regions(FRs) of a second (human) species.

A “human antibody” as used herein encompasses antibodies produced inhumans, antibodies produced in non-human animals that comprise humanimmunoglobulin genes, such as XENOMOUSE® mice, and antibodies selectedusing in vitro methods, such as phage display (Vaughan et al., 1996,Nat. Biotechnol., 14:309-314; Sheets et al., 1998, Proc. Natl. Acad.Sci. (USA), 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol.,227:381; Marks et al., 1991, J. Mol. Biol., 222:581), wherein theantibody repertoire is based on a human immunoglobulin sequence. Theterm “human antibody” denotes the genus of sequences that are humansequences. Thus, the term is not designating the process by which theantibody was created, but the genus of sequences that are relevant.

A “functional Fc region” possesses an “effector function” of a nativesequence Fc region. Exemplary “effector functions” include Fc receptorbinding; C1q binding; CDC; ADCC; phagocytosis; down regulation of cellsurface receptors (for example B cell receptor; BCR), etc. Such effectorfunctions generally require the Fc region to be combined with a bindingdomain (for example, an antibody variable domain) and can be assessedusing various assays.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. Nativesequence human Fc regions include a native sequence human IgG1 Fc region(non-A and A allotypes); native sequence human IgG2 Fc region; nativesequence human IgG3 Fc region; and native sequence human IgG4 Fc regionas well as naturally occurring variants thereof.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one aminoacid modification. In some embodiments, a “variant Fc region” comprisesan amino acid sequence which differs from that of a native sequence Fcregion by virtue of at least one amino acid modification, yet retains atleast one effector function of the native sequence Fc region. In someembodiments, the variant Fc region has at least one amino acidsubstitution compared to a native sequence Fc region or to the Fc regionof a parent polypeptide, for example, from about one to about ten aminoacid substitutions, and preferably, from about one to about five aminoacid substitutions in a native sequence Fc region or in the Fc region ofthe parent polypeptide. In some embodiments, the variant Fc regionherein will possess at least about 80% sequence identity with a nativesequence Fc region and/or with an Fc region of a parent polypeptide, atleast about 90% sequence identity therewith, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99% sequence identity therewith.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. In some embodiments, an FcγR is a native human FcR. Insome embodiments, an FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof those receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domainInhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. See, for example,Daeron, 1997, Annu. Rev. Immunol. 15:203-234. FcRs are reviewed, forexample, in Ravetch and Kinet, 1991, Annu. Rev. Immunol 9:457-92; Capelet al., 1994, Immunomethods, 4:25-34; and de Haas et al., 1995, J. Lab.Clin. Med. 126:330-41. Other FcRs, including those to be identified inthe future, are encompassed by the term “FcR” herein.

The term “Fc receptor” or “FcR” also includes the neonatal receptor,FcRn, which is responsible for the transfer of maternal IgGs to thefetus (Guyer et al., 1976, J. Immunol. 117:587 and Kim et al., 1994, J.Immunol. 24:249) and regulation of homeostasis of immunoglobulins.Methods of measuring binding to FcRn are known. See, for example, Ghetieand Ward, 1997, Immunol. Today, 18(12):592-598; Ghetie et al., 1997,Nat. Biotechnol., 15(7):637-640; Hinton et al., 2004, J. Biol. Chem.279(8):6213-6216; and WO 2004/92219 (Hinton et al.).

“Effector functions” refer to biological activities attributable to theFc region of an antibody, which vary with the antibody isotype. Examplesof antibody effector functions include: C1q binding and complementdependent cytotoxicity (CDC); Fc receptor binding; antibody-dependentcell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cellsurface receptors (for example B cell receptor); and B cell activation.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (for example NK cells, neutrophils,and macrophages) enable these cytotoxic effector cells to bindspecifically to an antigen-bearing target cell and subsequently kill thetarget cell with cytotoxins. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII,and FcγRIII. FcR expression on hematopoietic cells is summarized inTable 3 on page 464 of Ravetch and Kinet, 1991, Annu. Rev. Immunol9:457-92. To assess ADCC activity of a molecule of interest, an in vitroADCC assay, such as that described in U.S. Pat. Nos. 5,500,362,5,821,337, or 6,737,056 (Presta), may be performed. Useful effectorcells for such assays include PBMC and NK cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, for example, in an animal model such as that disclosed inClynes et al., 1998, Proc. Natl. Acad. Sci. (USA) 95:652-656. Additionalpolypeptide variants with altered Fc region amino acid sequences(polypeptides with a variant Fc region) and increased or decreased ADCCactivity are described, for example, in U.S. Pat. Nos. 7,923,538, and7,994,290.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass),which are bound to their cognate antigen. To assess complementactivation, a CDC assay, for example, as described in Gazzano-Santoro etal., 1996, J. Immunol. Methods 202:163, may be performed. Polypeptidevariants with altered Fc region amino acid sequences (polypeptides witha variant Fc region) and increased or decreased C1q binding capabilityare described, for example, in U.S. Pat. No. 6,194,551 B1, U.S. Pat.Nos. 7,923,538, 7,994,290, and WO 1999/51642. See also, for example,Idusogie et al., 2000, J. Immunol. 164: 4178-4184.

A polypeptide variant with “altered” FcR binding affinity or ADCCactivity is one which has either enhanced or diminished FcR bindingactivity and/or ADCC activity compared to a parent polypeptide or to apolypeptide comprising a native sequence Fc region. The polypeptidevariant which “displays increased binding” to an FcR binds at least oneFcR with better affinity than the parent polypeptide. The polypeptidevariant which “displays decreased binding” to an FcR, binds at least oneFcR with lower affinity than a parent polypeptide. Such variants whichdisplay decreased binding to an FcR may possess little or no appreciablebinding to an FcR, for example, 0-20% binding to the FcR compared to anative sequence IgG Fc region.

The polypeptide variant which “mediates antibody-dependent cell-mediatedcytotoxicity (ADCC) in the presence of human effector cells moreeffectively” than a parent antibody is one which in vitro or in vivo ismore effective at mediating ADCC, when the amounts of polypeptidevariant and parent antibody used in the assay are essentially the same.Generally, such variants will be identified using the in vitro ADCCassay as herein disclosed, but other assays or methods for determiningADCC activity, for example in an animal model etc., are contemplated.

The term “substantially similar” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between two ormore numeric values such that one of skill in the art would consider thedifference between the two or more values to be of little or nobiological and/or statistical significance within the context of thebiological characteristic measured by said value. In some embodimentsthe two or more substantially similar values differ by no more thanabout any one of 5%, 10%, 15%, 20%, 25%, or 50%.

The phrase “substantially different,” as used herein, denotes asufficiently high degree of difference between two numeric values suchthat one of skill in the art would consider the difference between thetwo values to be of statistical significance within the context of thebiological characteristic measured by said values. In some embodiments,the two substantially different numeric values differ by greater thanabout any one of 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%,90%, or 100%.

The phrase “substantially reduced,” as used herein, denotes asufficiently high degree of reduction between a numeric value and areference numeric value such that one of skill in the art would considerthe difference between the two values to be of statistical significancewithin the context of the biological characteristic measured by saidvalues. In some embodiments, the substantially reduced numeric values isreduced by greater than about any one of 10%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the reference value.

The term “leader sequence” refers to a sequence of amino acid residueslocated at the N-terminus of a polypeptide that facilitates secretion ofa polypeptide from a mammalian cell. A leader sequence can be cleavedupon export of the polypeptide from the mammalian cell, forming a matureprotein. Leader sequences can be natural or synthetic, and they can beheterologous or homologous to the protein to which they are attached.

A “native sequence” polypeptide comprises a polypeptide having the sameamino acid sequence as a polypeptide found in nature. Thus, a nativesequence polypeptide can have the amino acid sequence of naturallyoccurring polypeptide from any mammal. Such native sequence polypeptidecan be isolated from nature or can be produced by recombinant orsynthetic means. The term “native sequence” polypeptide specificallyencompasses naturally occurring truncated or secreted forms of thepolypeptide (for example, an extracellular domain sequence), naturallyoccurring variant forms (for example, alternatively spliced forms) andnaturally occurring allelic variants of the polypeptide.

A polypeptide “variant” means a biologically active polypeptide havingat least about 80% amino acid sequence identity with the native sequencepolypeptide after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Such variants include, for instance, polypeptides wherein oneor more amino acid residues are added, or deleted, at the N- orC-terminus of the polypeptide. In some embodiments, a variant will haveat least about 80% amino acid sequence identity. In some embodiments, avariant will have at least about 90% amino acid sequence identity. Insome embodiments, a variant will have at least about 95% amino acidsequence identity with the native sequence polypeptide.

As used herein, “percent (%) amino acid sequence identity” and“homology” with respect to a peptide, polypeptide or antibody sequenceare defined as the percentage of amino acid residues in a candidatesequence that are identical with the amino acid residues in the specificpeptide or polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.

An amino acid substitution may include but are not limited to thereplacement of one amino acid in a polypeptide with another amino acid.Exemplary substitutions are shown in Table 1. Amino acid substitutionsmay be introduced into an antibody of interest and the products screenedfor a desired activity, for example, retained/improved antigen binding,decreased immunogenicity, or improved ADCC or CDC.

TABLE 1 Original Residue Exemplary Substitutions Ala (A) Val; Leu; IleArg (R) Lys; Gln; Asn Asn (N) Gln; His; Asp, Lys; Arg Asp (D) Glu; AsnCys (C) Ser; Ala Gln (Q) Asn; Glu Glu (E) Asp; Gln Gly (G) Ala His (H)Asn; Gln; Lys; Arg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu (L)Norleucine; Ile; Val; Met; Ala; Phe Lys (K) Arg; Gln; Asn Met (M) Leu;Phe; Ile Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Pro (P) Ala Ser (S) ThrThr (T) Val; Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe; Thr; Ser Val (V)Ile; Leu; Met; Phe; Ala; Norleucine

Amino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

The term “vector” is used to describe a polynucleotide that can beengineered to contain a cloned polynucleotide or polynucleotides thatcan be propagated in a host cell. A vector can include one or more ofthe following elements: an origin of replication, one or more regulatorysequences (such as, for example, promoters and/or enhancers) thatregulate the expression of the polypeptide of interest, and/or one ormore selectable marker genes (such as, for example, antibioticresistance genes and genes that can be used in colorimetric assays, forexample, δ-galactosidase). The term “expression vector” refers to avector that is used to express a polypeptide of interest in a host cell.

A “host cell” refers to a cell that may be or has been a recipient of avector or isolated polynucleotide. Host cells may be prokaryotic cellsor eukaryotic cells. Exemplary eukaryotic cells include mammalian cells,such as primate or non-primate animal cells; fungal cells, such asyeast; plant cells; and insect cells. Nonlimiting exemplary mammaliancells include, but are not limited to, NSO cells, PER.C6® cells(Crucell), and 293 and CHO cells, and their derivatives, such as 293-6Eand DG44 cells, respectively. Host cells include progeny of a singlehost cell, and the progeny may not necessarily be completely identical(in morphology or in genomic DNA complement) to the original parent celldue to natural, accidental, or deliberate mutation. A host cell includescells transfected in vivo with a polynucleotide(s) a provided herein.

The term “isolated” as used herein refers to a molecule that has beenseparated from at least some of the components with which it istypically found in nature or produced. For example, a polypeptide isreferred to as “isolated” when it is separated from at least some of thecomponents of the cell in which it was produced. Where a polypeptide issecreted by a cell after expression, physically separating thesupernatant containing the polypeptide from the cell that produced it isconsidered to be “isolating” the polypeptide.

Similarly, a polynucleotide is referred to as “isolated” when it is notpart of the larger polynucleotide (such as, for example, genomic DNA ormitochondrial DNA, in the case of a DNA polynucleotide) in which it istypically found in nature, or is separated from at least some of thecomponents of the cell in which it was produced, for example, in thecase of an RNA polynucleotide. Thus, a DNA polynucleotide that iscontained in a vector inside a host cell may be referred to as“isolated”.

The terms “individual” or “subject” are used interchangeably herein torefer to an animal; for example a mammal. In some embodiments, methodsof treating mammals, including, but not limited to, humans, rodents,simians, felines, canines, equines, bovines, porcines, ovines, caprines,mammalian laboratory animals, mammalian farm animals, mammalian sportanimals, and mammalian pets, are provided. In some examples, an“individual” or “subject” refers to an individual or subject in need oftreatment for a disease or disorder. In some embodiments, the subject toreceive the treatment can be a patient, designating the fact that thesubject has been identified as having a disorder of relevance to thetreatment, or being at adequate risk of contracting the disorder.

A “disease” or “disorder” as used herein refers to a condition wheretreatment is needed and/or desired.

“Cancer” and “tumor,” as used herein, are interchangeable terms thatrefer to any abnormal cell or tissue growth or proliferation in ananimal. As used herein, the terms “cancer” and “tumor” encompass solidand hematological/lymphatic cancers and also encompass malignant,pre-malignant, and benign growth, such as dysplasia. Examples of cancerinclude but are not limited to, carcinoma, lymphoma, blastoma, sarcoma,and leukemia. More particular non-limiting examples of such cancersinclude kidney cancer (e.g., renal cell carcinoma, e.g., papillary renalcell carcinoma), squamous cell cancer, mesothelioma, teratoma,small-cell lung cancer, pituitary cancer, esophageal cancer,astrocytoma, soft tissue sarcoma, lung cancer (e.g., non-small cell lungcancer, adenocarcinoma of the lung, squamous carcinoma of the lung),cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer(e.g., stomach cancer), pancreatic cancer, cervical cancer, ovariancancer, liver cancer, bladder cancer, hepatoma, breast cancer, coloncancer, colorectal cancer, rectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, liver cancer, prostate cancer,vulval cancer, thyroid cancer, thymoma, hepatic carcinoma, brain cancer,glioma, glioblastoma, endometrial cancer, testis cancer,cholangiocarcinoma, cholangiosarcoma, gallbladder carcinoma, gastriccancer, melanoma (e.g., uveal melanoma), pheochromocytoma,paraganglioma, adenoid cystic carcinoma, and various types of head andneck cancer (e.g., squamous head and neck cancer).

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. “Treatment” as used herein, covers anyadministration or application of a therapeutic for disease in a mammal,including a human. For purposes of this disclosure, beneficial ordesired clinical results include, but are not limited to, any one ormore of: alleviation of one or more symptoms, diminishment of extent ofdisease, preventing or delaying spread (for example, metastasis, forexample metastasis to the lung or to the lymph node) of disease,preventing or delaying recurrence of disease, delay or slowing ofdisease progression, amelioration of the disease state, inhibiting thedisease or progression of the disease, inhibiting or slowing the diseaseor its progression, arresting its development, and remission (whetherpartial or total).

Also encompassed by “treatment” is a reduction of pathologicalconsequence of a proliferative disease. The methods provided hereincontemplate any one or more of these aspects of treatment. In-line withthe above, the term treatment does not require one-hundred percentremoval of all aspects of the disorder.

“Ameliorating” means a lessening or improvement of one or more symptomsas compared to not administering an anti-LILRB2 antibody. “Ameliorating”also includes shortening or reduction in duration of a symptom.

In the context of cancer, the term “treating” includes any or all of:inhibiting growth of cancer cells, inhibiting replication of cancercells, lessening of overall tumor burden and ameliorating one or moresymptoms associated with the disease.

The term “biological sample” means a quantity of a substance from aliving thing or formerly living thing. Such substances include, but arenot limited to, blood, (for example, whole blood), plasma, serum, urine,amniotic fluid, synovial fluid, endothelial cells, leukocytes,monocytes, other cells, organs, tissues, bone marrow, lymph nodes andspleen.

The term “control” refers to a composition known to not contain ananalyte (“negative control”) or to contain analyte (“positive control”).A positive control can comprise a known concentration of analyte.“Control,” “positive control,” and “calibrator” may be usedinterchangeably herein to refer to a composition comprising a knownconcentration of analyte. A “positive control” can be used to establishassay performance characteristics and is a useful indicator of theintegrity of reagents (for example, analytes).

“Predetermined cutoff” and “predetermined level” refer generally to anassay cutoff value that is used to assessdiagnostic/prognostic/therapeutic efficacy results by comparing theassay results against the predetermined cutoff/level, where thepredetermined cutoff/level already has been linked or associated withvarious clinical parameters (for example, severity of disease,progression, non-progression, improvement, etc.). While the presentdisclosure may provide exemplary predetermined levels, it is well-knownthat cutoff values may vary depending on the nature of the immunoassay(for example, antibodies employed, etc.). It further is well within theskill of one of ordinary skill in the art to adapt the disclosure hereinfor other immunoassays to obtain immunoassay-specific cutoff values forthose other immunoassays based on this disclosure. Whereas the precisevalue of the predetermined cutoff/level may vary between assays,correlations as described herein (if any) may be generally applicable.

The terms “inhibition” or “inhibit” refer to a decrease or cessation ofany phenotypic characteristic or to the decrease or cessation in theincidence, degree, or likelihood of that characteristic. To “reduce” or“inhibit” is to decrease, reduce or arrest an activity, function, and/oramount as compared to a reference. In some embodiments, by “reduce” or“inhibit” is meant the ability to cause an overall decrease of 20% orgreater. In some embodiments, by “reduce” or “inhibit” is meant theability to cause an overall decrease of 50% or greater. In someembodiments, by “reduce” or “inhibit” is meant the ability to cause anoverall decrease of 75%, 85%, 90%, 95%, or greater. In some embodiments,the amount noted above is inhibited or decreased over a period of time,relative to a control dose (such as a placebo) over the same period oftime. A “reference” as used herein, refers to any sample, standard, orlevel that is used for comparison purposes. A reference may be obtainedfrom a healthy and/or non-diseased sample. In some examples, a referencemay be obtained from an untreated sample. In some examples, a referenceis obtained from a non-diseased on non-treated sample of a subjectindividual. In some examples, a reference is obtained from one or morehealthy individuals who are not the subject or patient.

As used herein, “delaying development of a disease” means to defer,hinder, slow, retard, stabilize, suppress and/or postpone development ofthe disease (such as cancer). This delay can be of varying lengths oftime, depending on the history of the disease and/or individual beingtreated. As is evident to one skilled in the art, a sufficient orsignificant delay can, in effect, encompass prevention, in that theindividual does not develop the disease. For example, a late stagecancer, such as development of metastasis, may be delayed.

“Preventing,” as used herein, includes providing prophylaxis withrespect to the occurrence or recurrence of a disease in a subject thatmay be predisposed to the disease but has not yet been diagnosed withthe disease. Unless otherwise specified, the terms “reduce,” “inhibit,”or “prevent” do not denote or require complete prevention over all time.

As used herein, to “suppress” a function or activity is to reduce thefunction or activity when compared to otherwise same conditions exceptfor a condition or parameter of interest, or alternatively, as comparedto another condition. For example, an antibody which suppresses tumorgrowth reduces the rate of growth of the tumor compared to the rate ofgrowth of the tumor in the absence of the antibody.

A “therapeutically effective amount” of a substance/molecule, agonist orantagonist may vary according to factors such as the disease state, age,sex, and weight of the individual, and the ability of thesubstance/molecule, agonist or antagonist to elicit a desired responsein the individual. A therapeutically effective amount is also one inwhich any toxic or detrimental effects of the substance/molecule,agonist or antagonist are outweighed by the therapeutically beneficialeffects. A therapeutically effective amount may be delivered in one ormore administrations. A therapeutically effective amount refers to anamount effective, at dosages and for periods of time necessary, toachieve the desired therapeutic and/or prophylactic result.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically but not necessarily, since a prophylacticdose is used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

The terms “pharmaceutical formulation” and “pharmaceutical composition”refer to a preparation which is in such form as to permit the biologicalactivity of the active ingredient(s) to be effective, and which containsno additional components which are unacceptably toxic to a subject towhich the formulation would be administered. Such formulations may besterile.

A “pharmaceutically acceptable carrier” refers to a non-toxic solid,semisolid, or liquid filler, diluent, encapsulating material,formulation auxiliary, or carrier conventional in the art for use with atherapeutic agent that together comprise a “pharmaceutical composition”for administration to a subject. A pharmaceutically acceptable carrieris non-toxic to recipients at the dosages and concentrations employedand is compatible with other ingredients of the formulation. Thepharmaceutically acceptable carrier is appropriate for the formulationemployed.

A “sterile” formulation is aseptic or essentially free from livingmicroorganisms and their spores.

A “PD-1 therapy” encompasses any therapy that modulates PD-1 binding toPD-L1 and/or PD-L2. PD-1 therapies may, for example, directly interactwith PD-1 and/or PD-L1. In some embodiments, a PD-1 therapy includes amolecule that directly binds to and/or influences the activity of PD-1.In some embodiments, a PD-1 therapy includes a molecule that directlybinds to and/or influences the activity of PD-L1. Thus, an antibody thatbinds to PD-1 or PD-L1 and blocks the interaction of PD-1 to PD-L1 is aPD-1 therapeutic. When a desired subtype of PD-1 therapy is intended, itwill be designated by the phrase “PD-1 specific” for a therapy involvinga molecule that interacts directly with PD-1, or “PD-L1 specific” for amolecule that interacts directly with PD-L1, as appropriate. Unlessdesignated otherwise, all disclosure contained herein regarding PD-1therapy applies to PD-1 therapy generally, as well as PD-1 specificand/or PD-L1 specific therapies. Nonlimiting exemplary PD-1 therapiesinclude nivolumab (BMS-936558, MDX-1106, ONO-4538); pidilizumab,lambrolizumab/pembrolizumab (KEYTRUDA®, MK-3475); durvalumab; RG-7446;MSB-0010718C; AMP-224; BMS-936559 (an anti-PD-L1 antibody); AMP-514;MDX-1105; ANB-011; anti-LAG-3/PD-1; anti-PD-1 Ab (CoStim); anti-PD-1 Ab(Kadmon Pharm.); anti-PD-1 Ab (Immunovo); anti-TIM-3/PD-1 Ab(AnaptysBio); anti-PD-L1 Ab (CoStim/Novartis); MEDI-4736 (an anti-PD-L1antibody, Medimmune/AstraZeneca); RG7446/MPDL3280A (an anti-PD-L1antibody, Genentech/Roche); KD-033, PD-1 antagonist (Agenus); STI-A1010;STI-A1110; TSR-042; and other antibodies that are directed againstprogrammed death-1 (PD-1) or programmed death ligand 1 (PD-L1).

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive or sequentialadministration in any order.

The term “concurrently” is used herein to refer to administration of twoor more therapeutic agents, where at least part of the administrationoverlaps in time or where the administration of one therapeutic agentfalls within a short period of time relative to administration of theother therapeutic agent. For example, the two or more therapeutic agentsare administered with a time separation of no more than about aspecified number of minutes.

The term “sequentially” is used herein to refer to administration of twoor more therapeutic agents where the administration of one or moreagent(s) continues after discontinuing the administration of one or moreother agent(s). For example, administration of the two or moretherapeutic agents are administered with a time separation of more thanabout a specified number of minutes.

As used herein, “in conjunction with” refers to administration of onetreatment modality in addition to another treatment modality. As such,“in conjunction with” refers to administration of one treatment modalitybefore, during or after administration of the other treatment modalityto the individual.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

An “article of manufacture” is any manufacture (for example, a packageor container) or kit comprising at least one reagent, for example, amedicament for treatment of a disease or disorder (for example, cancer),or a probe for specifically detecting a biomarker described herein. Insome embodiments, the manufacture or kit is promoted, distributed, orsold as a unit for performing the methods described herein.

The terms “label” and “detectable label” mean a moiety attached to anantibody or its analyte to render a reaction (for example, binding)between the members of the specific binding pair, detectable. Thelabeled member of the specific binding pair is referred to as“detectably labeled.” Thus, the term “labeled binding protein” refers toa protein with a label incorporated that provides for the identificationof the binding protein. In some embodiments, the label is a detectablemarker that can produce a signal that is detectable by visual orinstrumental means, for example, incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (for example, streptavidin containing afluorescent marker or enzymatic activity that can be detected by opticalor colorimetric methods). Examples of labels for polypeptides include,but are not limited to, the following: radioisotopes or radionuclides(for example, ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁸⁸Ho,or ¹⁵³Sm); chromogens, fluorescent labels (for example, FITC, rhodamine,lanthanide phosphors), enzymatic labels (for example, horseradishperoxidase, luciferase, alkaline phosphatase); chemiluminescent markers;biotinyl groups; predetermined polypeptide epitopes recognized by asecondary reporter (for example, leucine zipper pair sequences, bindingsites for secondary antibodies, metal binding domains, epitope tags);and magnetic agents, such as gadolinium chelates. Representativeexamples of labels commonly employed for immunoassays include moietiesthat produce light, for example, acridinium compounds, and moieties thatproduce fluorescence, for example, fluorescein. In this regard, themoiety itself may not be detectably labeled but may become detectableupon reaction with yet another moiety.

The term “conjugate” refers to an antibody that is chemically linked toa second chemical moiety, such as a therapeutic or cytotoxic agent. Theterm “agent” includes a chemical compound, a mixture of chemicalcompounds, a biological macromolecule, or an extract made frombiological materials. In some embodiments, the therapeutic or cytotoxicagents include, but are not limited to, pertussis toxin, taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. When employed in the context of an immunoassay, theconjugate antibody may be a detectably labeled antibody used as thedetection antibody.

II. Anti-LILRB2 Antibodies

Novel antibodies directed against LILRB2 are provided. Anti-LILRB2antibodies include, but are not limited to, humanized antibodies,chimeric antibodies, mouse antibodies, human antibodies, and antibodiescomprising the heavy chain and/or light chain CDRs discussed herein. Insome embodiments, an isolated antibody that binds to LILRB2 is provided.In some embodiments, a monoclonal antibody that binds to LILRB2 isprovided.

In some aspects, an anti-LILRB2 antibody comprises at least one, two,three, four, five, or six CDRs selected from (a) CDR-H1 comprising theamino acid sequence of SEQ ID NO: 15; (b) CDR-H2 comprising the aminoacid sequence of SEQ ID NO: 16; (c) CDR-H3 comprising the amino acidsequence of SEQ ID NO: 17; (d) CDR-L1 comprising the amino acid sequenceof SEQ ID NO: 18; (e) CDR-L2 comprising the amino acid sequence of SEQID NO: 19; and (f) CDR-L3 comprising the amino acid sequence of SEQ IDNO: 20.

In some aspects, an anti-LILRB2 antibody comprises at least one, two,three, four, five, or six CDRs selected from (a) CDR-H1 comprising theamino acid sequence of SEQ ID NO: 95; (b) CDR-H2 comprising the aminoacid sequence of SEQ ID NO: 96; (c) CDR-H3 comprising the amino acidsequence of SEQ ID NO: 97; (d) CDR-L1 comprising the amino acid sequenceof SEQ ID NO: 98; (e) CDR-L2 comprising the amino acid sequence of SEQID NO: 99; and (f) CDR-L3 comprising the amino acid sequence of SEQ IDNO: 100.

In some aspects, an anti-LILRB2 antibody comprises at least one, two,three, four, five, or six CDRs selected from (a) CDR-H1 comprising theamino acid sequence of SEQ ID NO: 105; (b) CDR-H2 comprising the aminoacid sequence of SEQ ID NO: 106; (c) CDR-H3 comprising the amino acidsequence of SEQ ID NO: 107; (d) CDR-L1 comprising the amino acidsequence of SEQ ID NO: 108; (e) CDR-L2 comprising the amino acidsequence of SEQ ID NO: 109; and (f) CDR-L3 comprising the amino acidsequence of SEQ ID NO: 110.

In some aspects, an anti-LILRB2 antibody comprises at least one, two,three, four, five, or six CDRs selected from (a) CDR-H1 comprising theamino acid sequence of SEQ ID NO: 115; (b) CDR-H2 comprising the aminoacid sequence of SEQ ID NO: 116; (c) CDR-H3 comprising the amino acidsequence of SEQ ID NO: 117; (d) CDR-L1 comprising the amino acidsequence of SEQ ID NO: 118; (e) CDR-L2 comprising the amino acidsequence of SEQ ID NO: 119; and (f) CDR-L3 comprising the amino acidsequence of SEQ ID NO: 120.

In some embodiments, an anti-LILRB2 antibody is provided that competeswith an anti-LILRB2 antibody described herein. In some embodiments, anantibody that competes for binding (i.e., cross-competes) with any ofthe antibodies provided herein can be made and/or used.

In some embodiments, the anti-LILRB2 antibody comprises at least one, atleast two, or all three heavy chain CDR sequences selected from (a)CDR-H1 comprising the amino acid sequence of SEQ ID NO: 15; (b) CDR-H2comprising the amino acid sequence of SEQ ID NO: 16; and (c) CDR-H3comprising the amino acid sequence of SEQ ID NO: 17.

In some embodiments, the anti-LILRB2 antibody comprises at least one, atleast two, or all three light chain CDR sequences selected from (a)CDR-L1 comprising the amino acid sequence of SEQ ID NO: 18; (b) CDR-L2comprising the amino acid sequence of SEQ ID NO: 19; and (c) CDR-L3comprising the amino acid sequence of SEQ ID NO: 20.

In some embodiments, the anti-LILRB2 antibody comprises at least one, atleast two, or all three heavy chain CDR sequences selected from (a)CDR-H1 comprising the amino acid sequence of SEQ ID NO: 95; (b) CDR-H2comprising the amino acid sequence of SEQ ID NO: 96; and (c) CDR-H3comprising the amino acid sequence of SEQ ID NO: 97.

In some embodiments, the anti-LILRB2 antibody comprises at least one, atleast two, or all three light chain CDR sequences selected from (a)CDR-L1 comprising the amino acid sequence of SEQ ID NO: 98; (b) CDR-L2comprising the amino acid sequence of SEQ ID NO: 99; and (c) CDR-L3comprising the amino acid sequence of SEQ ID NO: 100.

In some embodiments, the anti-LILRB2 antibody comprises at least one, atleast two, or all three heavy chain CDR sequences selected from (a)CDR-H1 comprising the amino acid sequence of SEQ ID NO: 105; (b) CDR-H2comprising the amino acid sequence of SEQ ID NO: 106; and (c) CDR-H3comprising the amino acid sequence of SEQ ID NO: 107.

In some embodiments, the anti-LILRB2 antibody comprises at least one, atleast two, or all three light chain CDR sequences selected from (a)CDR-L1 comprising the amino acid sequence of SEQ ID NO: 108;

(b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 109; and (c)CDR-L3 comprising the amino acid sequence of SEQ ID NO: 110.

In some embodiments, the anti-LILRB2 antibody comprises at least one, atleast two, or all three heavy chain CDR sequences selected from (a)CDR-H1 comprising the amino acid sequence of SEQ ID NO: 115; (b) CDR-H2comprising the amino acid sequence of SEQ ID NO: 116; and (c) CDR-H3comprising the amino acid sequence of SEQ ID NO: 117.

In some embodiments, the anti-LILRB2 antibody comprises at least one, atleast two, or all three light chain CDR sequences selected from (a)CDR-L1 comprising the amino acid sequence of SEQ ID NO: 118; (b) CDR-L2comprising the amino acid sequence of SEQ ID NO: 119; and (c) CDR-L3comprising the amino acid sequence of SEQ ID NO: 120.

In some embodiments, any of the six CDRs provided herein can be combinedas subparts with any of the other CDRs provided herein, for a total ofsix CDRs in a construct. Thus, in some embodiments, two CDRs from afirst antibody (for example, CDR-H1 and CDR-H2) can be combined withfour CDRs from a second antibody (CDR-H3, CDR-L1, CDR-L2, and CDR-L3).In some embodiments, two or fewer residues in one or more of the CDRscan be replaced to obtain a variant thereof. In some embodiments, two orfewer residues can be replaced in 1, 2, 3, 4, 5, or 6 of the CDRs.

In particular embodiments, an anti-LILRB2 antibody comprises a variableheavy chain (V_(H)) domain sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO: 13. In some embodiments, a V_(H) sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity contains substitutions (for example, conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-LILRB2 antibody comprising that sequence retainsthe ability to bind to LILRB2. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, and/or deleted in SEQ IDNO: 13. In some embodiments, substitutions, insertions, or deletionsoccur in regions outside the CDRs (that is, in the FRs). Optionally, theanti-LILRB2 antibody comprises the V_(H) sequence of SEQ ID NO: 13,including post-translational modifications of that sequence.

In some embodiments, the V_(H) comprises: (a) CDR-H1 comprising theamino acid sequence of SEQ ID NO: 15; (b) CDR-H2 comprising the aminoacid sequence of SEQ ID NO: 16; and (c) CDR-H3 comprising the amino acidsequence of SEQ ID NO: 17.

In some embodiments, an anti-LILRB2 antibody is provided, wherein theantibody comprises a variable light chain (V_(L)) domain having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 14. In someembodiments, a V_(L) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity contains substitutions (for example,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-LILRB2 antibody comprising that sequenceretains the ability to bind to LILRB2. In some embodiments, a total of 1to 10 amino acids have been substituted, inserted, and/or deleted in SEQID NO: 14. In some embodiments, the substitutions, insertions, ordeletions occur in regions outside the CDRs (that is, in the FRs).Optionally, the anti-LILRB2 antibody comprises the V_(L) sequence of SEQID NO: 14, including post-translational modifications of that sequence.

In some embodiments, the V_(L) comprises: (a) CDR-L1 comprising theamino acid sequence of SEQ ID NO: 18; (b) CDR-L2 comprising the aminoacid sequence of SEQ ID NO: 19; and (c) CDR-L3 comprising the amino acidsequence of SEQ ID NO: 20.

In some embodiments, an anti-LILRB2 antibody comprises a V_(H) domainsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:13 and a V_(L) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ IDNO: 14. In some embodiments, a V_(H) domain sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (for example, conservative substitutions), insertions, ordeletions relative to the reference sequence, and a V_(L) domainsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity contains substitutions (for example, conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-LILRB2 antibody comprising that sequence retainsthe ability to bind to LILRB2. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, and/or deleted in SEQ IDNO: 13. In some embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted, and/or deleted in SEQ ID NO: 14. In someembodiments, substitutions, insertions, or deletions occur in regionsoutside the CDRs (that is, in the FRs). Optionally, the anti-LILRB2antibody comprises the V_(H) domain sequence in SEQ ID NO: 13 and theV_(L) domain sequence of SEQ ID NO: 14, including post-translationalmodifications of one or both sequence.

In some embodiments, an anti-LILRB2 antibody comprises a V_(H) domain asin any of the embodiments provided herein, and a V_(L) domain as in anyof the embodiments provided herein. In some embodiments, the antibodycomprises the V_(H) and V_(L) domain sequences of SEQ ID NO: 13 and SEQID NO: 14, respectively, including post-translational modifications ofthose sequences.

In some embodiments, an anti-LILRB2 antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 11 and a light chaincomprising the amino acid sequence of SEQ ID NO: 12.

In particular embodiments, an anti-LILRB2 antibody comprises a variableheavy chain (V_(H)) domain sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO: 53. In some embodiments, a V_(H) sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity contains substitutions (for example, conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-LILRB2 antibody comprising that sequence retainsthe ability to bind to LILRB2. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, and/or deleted in SEQ IDNO: 53. In some embodiments, substitutions, insertions, or deletionsoccur in regions outside the CDRs (that is, in the FRs). Optionally, theanti-LILRB2 antibody comprises the V_(H) sequence of SEQ ID NO: 53,including post-translational modifications of that sequence.

In some embodiments, the V_(H) comprises: (a) CDR-H1 comprising theamino acid sequence of SEQ ID NO: 15; (b) CDR-H2 comprising the aminoacid sequence of SEQ ID NO: 16; and (c) CDR-H3 comprising the amino acidsequence of SEQ ID NO: 17.

In some embodiments, an anti-LILRB2 antibody is provided, wherein theantibody comprises a variable light chain (V_(L)) domain having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 54. In someembodiments, a V_(L) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity contains substitutions (for example,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-LILRB2 antibody comprising that sequenceretains the ability to bind to LILRB2. In some embodiments, a total of 1to 10 amino acids have been substituted, inserted, and/or deleted in SEQID NO: 54. In some embodiments, the substitutions, insertions, ordeletions occur in regions outside the CDRs (that is, in the FRs).Optionally, the anti-LILRB2 antibody comprises the V_(L) sequence of SEQID NO: 54, including post-translational modifications of that sequence.

In some embodiments, the V_(L) comprises: (a) CDR-L1 comprising theamino acid sequence of SEQ ID NO: 18; (b) CDR-L2 comprising the aminoacid sequence of SEQ ID NO: 19; and (c) CDR-L3 comprising the amino acidsequence of SEQ ID NO: 20.

In some embodiments, an anti-LILRB2 antibody comprises a V_(H) domainsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:53 and a V_(L) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ IDNO: 54. In some embodiments, a V_(H) domain sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (for example, conservative substitutions), insertions, ordeletions relative to the reference sequence, and a V_(L) domainsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity contains substitutions (for example, conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-LILRB2 antibody comprising that sequence retainsthe ability to bind to LILRB2. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, and/or deleted in SEQ IDNO: 53. In some embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted, and/or deleted in SEQ ID NO: 54. In someembodiments, substitutions, insertions, or deletions occur in regionsoutside the CDRs (that is, in the FRs). Optionally, the anti-LILRB2antibody comprises the V_(H) domain sequence in SEQ ID NO: 53 and theV_(L) domain sequence of SEQ ID NO: 54, including post-translationalmodifications of one or both sequence.

In some embodiments, an anti-LILRB2 antibody comprises a V_(H) domain asin any of the embodiments provided herein, and a V_(L) domain as in anyof the embodiments provided herein. In some embodiments, the antibodycomprises the V_(H) and V_(L) domain sequences of SEQ ID NO: 53 and SEQID NO: 54, respectively, including post-translational modifications ofthose sequences.

In some embodiments, an anti-LILRB2 antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 51 and a light chaincomprising the amino acid sequence of SEQ ID NO: 52.

In particular embodiments, an anti-LILRB2 antibody comprises a variableheavy chain (V_(H)) domain sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO: 63. In some embodiments, a V_(H) sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity contains substitutions (for example, conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-LILRB2 antibody comprising that sequence retainsthe ability to bind to LILRB2. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, and/or deleted in SEQ IDNO: 63. In some embodiments, substitutions, insertions, or deletionsoccur in regions outside the CDRs (that is, in the FRs). Optionally, theanti-LILRB2 antibody comprises the V_(H) sequence of SEQ ID NO: 63,including post-translational modifications of that sequence.

In some embodiments, the V_(H) comprises: (a) CDR-H1 comprising theamino acid sequence of SEQ ID NO: 15; (b) CDR-H2 comprising the aminoacid sequence of SEQ ID NO: 16; and (c) CDR-H3 comprising the amino acidsequence of SEQ ID NO: 17.

In some embodiments, an anti-LILRB2 antibody is provided, wherein theantibody comprises a variable light chain (V_(L)) domain having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 64. In someembodiments, a V_(L) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity contains substitutions (for example,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-LILRB2 antibody comprising that sequenceretains the ability to bind to LILRB2. In some embodiments, a total of 1to 10 amino acids have been substituted, inserted, and/or deleted in SEQID NO: 64. In some embodiments, the substitutions, insertions, ordeletions occur in regions outside the CDRs (that is, in the FRs).Optionally, the anti-LILRB2 antibody comprises the V_(L) sequence of SEQID NO: 64, including post-translational modifications of that sequence.

In some embodiments, the V_(L) comprises: (a) CDR-L1 comprising theamino acid sequence of SEQ ID NO: 18; (b) CDR-L2 comprising the aminoacid sequence of SEQ ID NO: 19; and (c) CDR-L3 comprising the amino acidsequence of SEQ ID NO: 20.

In some embodiments, an anti-LILRB2 antibody comprises a V_(H) domainsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:63 and a V_(L) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ IDNO: 64. In some embodiments, a V_(H) domain sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (for example, conservative substitutions), insertions, ordeletions relative to the reference sequence, and a V_(L) domainsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity contains substitutions (for example, conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-LILRB2 antibody comprising that sequence retainsthe ability to bind to LILRB2. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, and/or deleted in SEQ IDNO: 63. In some embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted, and/or deleted in SEQ ID NO: 64. In someembodiments, substitutions, insertions, or deletions occur in regionsoutside the CDRs (that is, in the FRs). Optionally, the anti-LILRB2antibody comprises the V_(H) domain sequence in SEQ ID NO: 63 and theV_(L) domain sequence of SEQ ID NO: 64, including post-translationalmodifications of one or both sequence.

In some embodiments, an anti-LILRB2 antibody comprises a V_(H) domain asin any of the embodiments provided herein, and a V_(L) domain as in anyof the embodiments provided herein. In some embodiments, the antibodycomprises the V_(H) and V_(L) domain sequences of SEQ ID NO: 63 and SEQID NO: 64, respectively, including post-translational modifications ofthose sequences.

In some embodiments, an anti-LILRB2 antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 61 and a light chaincomprising the amino acid sequence of SEQ ID NO: 62.

In particular embodiments, an anti-LILRB2 antibody comprises a variableheavy chain (V_(H)) domain sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO: 73. In some embodiments, a V_(H) sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity contains substitutions (for example, conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-LILRB2 antibody comprising that sequence retainsthe ability to bind to LILRB2. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, and/or deleted in SEQ IDNO: 73. In some embodiments, substitutions, insertions, or deletionsoccur in regions outside the CDRs (that is, in the FRs). Optionally, theanti-LILRB2 antibody comprises the V_(H) sequence of SEQ ID NO: 73,including post-translational modifications of that sequence.

In some embodiments, the V_(H) comprises: (a) CDR-H1 comprising theamino acid sequence of SEQ ID NO: 15; (b) CDR-H2 comprising the aminoacid sequence of SEQ ID NO: 16; and (c) CDR-H3 comprising the amino acidsequence of SEQ ID NO: 17.

In some embodiments, an anti-LILRB2 antibody is provided, wherein theantibody comprises a variable light chain (V_(L)) domain having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 74. In someembodiments, a V_(L) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity contains substitutions (for example,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-LILRB2 antibody comprising that sequenceretains the ability to bind to LILRB2. In some embodiments, a total of 1to 10 amino acids have been substituted, inserted, and/or deleted in SEQID NO: 74. In some embodiments, the substitutions, insertions, ordeletions occur in regions outside the CDRs (that is, in the FRs).Optionally, the anti-LILRB2 antibody comprises the V_(L) sequence of SEQID NO: 74, including post-translational modifications of that sequence.

In some embodiments, the V_(L) comprises: (a) CDR-L1 comprising theamino acid sequence of SEQ ID NO: 18; (b) CDR-L2 comprising the aminoacid sequence of SEQ ID NO: 19; and (c) CDR-L3 comprising the amino acidsequence of SEQ ID NO: 20.

In some embodiments, an anti-LILRB2 antibody comprises a V_(H) domainsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:73 and a V_(L) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ IDNO: 74. In some embodiments, a V_(H) domain sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (for example, conservative substitutions), insertions, ordeletions relative to the reference sequence, and a V_(L) domainsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity contains substitutions (for example, conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-LILRB2 antibody comprising that sequence retainsthe ability to bind to LILRB2. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, and/or deleted in SEQ IDNO: 73. In some embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted, and/or deleted in SEQ ID NO: 74. In someembodiments, substitutions, insertions, or deletions occur in regionsoutside the CDRs (that is, in the FRs). Optionally, the anti-LILRB2antibody comprises the V_(H) domain sequence in SEQ ID NO: 73 and theV_(L) domain sequence of SEQ ID NO: 74, including post-translationalmodifications of one or both sequence.

In some embodiments, an anti-LILRB2 antibody comprises a V_(H) domain asin any of the embodiments provided herein, and a V_(L) domain as in anyof the embodiments provided herein. In some embodiments, the antibodycomprises the V_(H) and V_(L) domain sequences of SEQ ID NO: 73 and SEQID NO: 74, respectively, including post-translational modifications ofthose sequences.

In some embodiments, an anti-LILRB2 antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 71 and a light chaincomprising the amino acid sequence of SEQ ID NO: 72.

In particular embodiments, an anti-LILRB2 antibody comprises a variableheavy chain (V_(H)) domain sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO: 83. In some embodiments, a V_(H) sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity contains substitutions (for example, conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-LILRB2 antibody comprising that sequence retainsthe ability to bind to LILRB2. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, and/or deleted in SEQ IDNO: 83. In some embodiments, substitutions, insertions, or deletionsoccur in regions outside the CDRs (that is, in the FRs). Optionally, theanti-LILRB2 antibody comprises the V_(H) sequence of SEQ ID NO: 83,including post-translational modifications of that sequence.

In some embodiments, the V_(H) comprises: (a) CDR-H1 comprising theamino acid sequence of SEQ ID NO: 15; (b) CDR-H2 comprising the aminoacid sequence of SEQ ID NO: 16; and (c) CDR-H3 comprising the amino acidsequence of SEQ ID NO: 17.

In some embodiments, an anti-LILRB2 antibody is provided, wherein theantibody comprises a variable light chain (V_(L)) domain having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 84. In someembodiments, a V_(L) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity contains substitutions (for example,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-LILRB2 antibody comprising that sequenceretains the ability to bind to LILRB2. In some embodiments, a total of 1to 10 amino acids have been substituted, inserted, and/or deleted in SEQID NO: 84. In some embodiments, the substitutions, insertions, ordeletions occur in regions outside the CDRs (that is, in the FRs).Optionally, the anti-LILRB2 antibody comprises the V_(L) sequence of SEQID NO: 84, including post-translational modifications of that sequence.

In some embodiments, the V_(L) comprises: (a) CDR-L1 comprising theamino acid sequence of SEQ ID NO: 18; (b) CDR-L2 comprising the aminoacid sequence of SEQ ID NO: 19; and (c) CDR-L3 comprising the amino acidsequence of SEQ ID NO: 20.

In some embodiments, an anti-LILRB2 antibody comprises a V_(H) domainsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:83 and a V_(L) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ IDNO: 84. In some embodiments, a V_(H) domain sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (for example, conservative substitutions), insertions, ordeletions relative to the reference sequence, and a V_(L) domainsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity contains substitutions (for example, conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-LILRB2 antibody comprising that sequence retainsthe ability to bind to LILRB2. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, and/or deleted in SEQ IDNO: 83. In some embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted, and/or deleted in SEQ ID NO: 84. In someembodiments, substitutions, insertions, or deletions occur in regionsoutside the CDRs (that is, in the FRs). Optionally, the anti-LILRB2antibody comprises the V_(H) domain sequence in SEQ ID NO: 83 and theV_(L) domain sequence of SEQ ID NO: 84, including post-translationalmodifications of one or both sequence.

In some embodiments, an anti-LILRB2 antibody comprises a V_(H) domain asin any of the embodiments provided herein, and a V_(L) domain as in anyof the embodiments provided herein. In some embodiments, the antibodycomprises the V_(H) and V_(L) domain sequences of SEQ ID NO: 83 and SEQID NO: 84, respectively, including post-translational modifications ofthose sequences.

In some embodiments, an anti-LILRB2 antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 81 and a light chaincomprising the amino acid sequence of SEQ ID NO: 82.

In particular embodiments, an anti-LILRB2 antibody comprises a variableheavy chain (V_(H)) domain sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO: 93. In some embodiments, a V_(H) sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity contains substitutions (for example, conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-LILRB2 antibody comprising that sequence retainsthe ability to bind to LILRB2. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, and/or deleted in SEQ IDNO: 93. In some embodiments, substitutions, insertions, or deletionsoccur in regions outside the CDRs (that is, in the FRs). Optionally, theanti-LILRB2 antibody comprises the V_(H) sequence of SEQ ID NO: 93,including post-translational modifications of that sequence.

In some embodiments, the V_(H) comprises: (a) CDR-H1 comprising theamino acid sequence of SEQ ID NO: 95; (b) CDR-H2 comprising the aminoacid sequence of SEQ ID NO: 96; and (c) CDR-H3 comprising the amino acidsequence of SEQ ID NO: 97.

In some embodiments, an anti-LILRB2 antibody is provided, wherein theantibody comprises a variable light chain (V_(L)) domain having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 94. In someembodiments, a V_(L) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity contains substitutions (for example,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-LILRB2 antibody comprising that sequenceretains the ability to bind to LILRB2. In some embodiments, a total of 1to 10 amino acids have been substituted, inserted, and/or deleted in SEQID NO: 94. In some embodiments, the substitutions, insertions, ordeletions occur in regions outside the CDRs (that is, in the FRs).Optionally, the anti-LILRB2 antibody comprises the V_(L) sequence of SEQID NO: 94, including post-translational modifications of that sequence.

In some embodiments, the V_(L) comprises: (a) CDR-L1 comprising theamino acid sequence of SEQ ID NO: 98; (b) CDR-L2 comprising the aminoacid sequence of SEQ ID NO: 99; and (c) CDR-L3 comprising the amino acidsequence of SEQ ID NO: 100.

In some embodiments, an anti-LILRB2 antibody comprises a V_(H) domainsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:93 and a V_(L) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ IDNO: 94. In some embodiments, a V_(H) domain sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (for example, conservative substitutions), insertions, ordeletions relative to the reference sequence, and a V_(L) domainsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity contains substitutions (for example, conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-LILRB2 antibody comprising that sequence retainsthe ability to bind to LILRB2. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, and/or deleted in SEQ IDNO: 93. In some embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted, and/or deleted in SEQ ID NO: 94. In someembodiments, substitutions, insertions, or deletions occur in regionsoutside the CDRs (that is, in the FRs). Optionally, the anti-LILRB2antibody comprises the V_(H) domain sequence in SEQ ID NO: 93 and theV_(L) domain sequence of SEQ ID NO: 94, including post-translationalmodifications of one or both sequence.

In some embodiments, an anti-LILRB2 antibody comprises a V_(H) domain asin any of the embodiments provided herein, and a V_(L) domain as in anyof the embodiments provided herein. In some embodiments, the antibodycomprises the V_(H) and V_(L) domain sequences of SEQ ID NO: 93 and SEQID NO: 94, respectively, including post-translational modifications ofthose sequences.

In some embodiments, an anti-LILRB2 antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 91 and a light chaincomprising the amino acid sequence of SEQ ID NO: 92.

In particular embodiments, an anti-LILRB2 antibody comprises a variableheavy chain (V_(H)) domain sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO: 103. In some embodiments, a V_(H) sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity contains substitutions (for example, conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-LILRB2 antibody comprising that sequence retainsthe ability to bind to LILRB2. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, and/or deleted in SEQ IDNO: 103. In some embodiments, substitutions, insertions, or deletionsoccur in regions outside the CDRs (that is, in the FRs). Optionally, theanti-LILRB2 antibody comprises the V_(H) sequence of SEQ ID NO: 103,including post-translational modifications of that sequence.

In some embodiments, the V_(H) comprises: (a) CDR-H1 comprising theamino acid sequence of SEQ ID NO: 105; (b) CDR-H2 comprising the aminoacid sequence of SEQ ID NO: 106; and (c) CDR-H3 comprising the aminoacid sequence of SEQ ID NO: 107.

In some embodiments, an anti-LILRB2 antibody is provided, wherein theantibody comprises a variable light chain (V_(L)) domain having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 104. In someembodiments, a V_(L) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity contains substitutions (for example,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-LILRB2 antibody comprising that sequenceretains the ability to bind to LILRB2. In some embodiments, a total of 1to 10 amino acids have been substituted, inserted, and/or deleted in SEQID NO: 104. In some embodiments, the substitutions, insertions, ordeletions occur in regions outside the CDRs (that is, in the FRs).Optionally, the anti-LILRB2 antibody comprises the V_(L) sequence of SEQID NO: 104, including post-translational modifications of that sequence.

In some embodiments, the V_(L) comprises: (a) CDR-L1 comprising theamino acid sequence of SEQ ID NO: 108; (b) CDR-L2 comprising the aminoacid sequence of SEQ ID NO: 109; and (c) CDR-L3 comprising the aminoacid sequence of SEQ ID NO: 110.

In some embodiments, an anti-LILRB2 antibody comprises a V_(H) domainsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:103 and a V_(L) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ IDNO: 104. In some embodiments, a V_(H) domain sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (for example, conservative substitutions), insertions, ordeletions relative to the reference sequence, and a V_(L) domainsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity contains substitutions (for example, conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-LILRB2 antibody comprising that sequence retainsthe ability to bind to LILRB2. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, and/or deleted in SEQ IDNO: 103. In some embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted, and/or deleted in SEQ ID NO: 104. In someembodiments, substitutions, insertions, or deletions occur in regionsoutside the CDRs (that is, in the FRs). Optionally, the anti-LILRB2antibody comprises the V_(H) domain sequence in SEQ ID NO: 103 and theV_(L) domain sequence of SEQ ID NO: 104, including post-translationalmodifications of one or both sequence.

In some embodiments, an anti-LILRB2 antibody comprises a V_(H) domain asin any of the embodiments provided herein, and a V_(L) domain as in anyof the embodiments provided herein. In some embodiments, the antibodycomprises the V_(H) and V_(L) domain sequences of SEQ ID NO: 103 and SEQID NO: 104, respectively, including post-translational modifications ofthose sequences.

In some embodiments, an anti-LILRB2 antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 101 and a light chaincomprising the amino acid sequence of SEQ ID NO: 102.

In particular embodiments, an anti-LILRB2 antibody comprises a variableheavy chain (V_(H)) domain sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO: 113. In some embodiments, a V_(H) sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity contains substitutions (for example, conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-LILRB2 antibody comprising that sequence retainsthe ability to bind to LILRB2. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, and/or deleted in SEQ IDNO: 113. In some embodiments, substitutions, insertions, or deletionsoccur in regions outside the CDRs (that is, in the FRs). Optionally, theanti-LILRB2 antibody comprises the V_(H) sequence of SEQ ID NO: 113,including post-translational modifications of that sequence.

In some embodiments, the V_(H) comprises: (a) CDR-H1 comprising theamino acid sequence of SEQ ID NO: 115; (b) CDR-H2 comprising the aminoacid sequence of SEQ ID NO: 116; and (c) CDR-H3 comprising the aminoacid sequence of SEQ ID NO: 117.

In some embodiments, an anti-LILRB2 antibody is provided, wherein theantibody comprises a variable light chain (V_(L)) domain having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 114. In someembodiments, a V_(L) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity contains substitutions (for example,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-LILRB2 antibody comprising that sequenceretains the ability to bind to LILRB2. In some embodiments, a total of 1to 10 amino acids have been substituted, inserted, and/or deleted in SEQID NO: 114. In some embodiments, the substitutions, insertions, ordeletions occur in regions outside the CDRs (that is, in the FRs).Optionally, the anti-LILRB2 antibody comprises the V_(L) sequence of SEQID NO: 114, including post-translational modifications of that sequence.

In some embodiments, the V_(L) comprises: (a) CDR-L1 comprising theamino acid sequence of SEQ ID NO: 118; (b) CDR-L2 comprising the aminoacid sequence of SEQ ID NO: 119; and (c) CDR-L3 comprising the aminoacid sequence of SEQ ID NO: 120.

In some embodiments, an anti-LILRB2 antibody comprises a V_(H) domainsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:113 and a V_(L) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ IDNO: 114. In some embodiments, a V_(H) domain sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (for example, conservative substitutions), insertions, ordeletions relative to the reference sequence, and a V_(L) domainsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity contains substitutions (for example, conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-LILRB2 antibody comprising that sequence retainsthe ability to bind to LILRB2. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, and/or deleted in SEQ IDNO: 113. In some embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted, and/or deleted in SEQ ID NO: 114. In someembodiments, substitutions, insertions, or deletions occur in regionsoutside the CDRs (that is, in the FRs). Optionally, the anti-LILRB2antibody comprises the V_(H) domain sequence in SEQ ID NO: 113 and theV_(L) domain sequence of SEQ ID NO: 114, including post-translationalmodifications of one or both sequence.

In some embodiments, an anti-LILRB2 antibody comprises a V_(H) domain asin any of the embodiments provided herein, and a V_(L) domain as in anyof the embodiments provided herein. In some embodiments, the antibodycomprises the V_(H) and V_(L) domain sequences of SEQ ID NO: 113 and SEQID NO: 114, respectively, including post-translational modifications ofthose sequences.

In some embodiments, an anti-LILRB2 antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 111 and a light chaincomprising the amino acid sequence of SEQ ID NO: 112.

In some aspects, an anti-LILRB2 antibody comprises at least one, two,three, four, five, or six CDRs selected from (a) CDR-H1 comprising theamino acid sequence of SEQ ID NO: 5; (b) CDR-H2 comprising the aminoacid sequence of SEQ ID NO: 6; (c) CDR-H3 comprising the amino acidsequence of SEQ ID NO: 7; (d) CDR-L1 comprising the amino acid sequenceof SEQ ID NO: 8; (e) CDR-L2 comprising the amino acid sequence of SEQ IDNO: 9; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:10.

In some embodiments, an anti-LILRB2 antibody is provided that competeswith an anti-LILRB2 antibody described herein. In some embodiments, anantibody that competes for binding (i.e., cross-competes) with any ofthe antibodies provided herein can be made and/or used.

In some embodiments, the anti-LILRB2 antibody comprises at least one, atleast two, or all three heavy chain CDR sequences selected from (a)CDR-H1 comprising the amino acid sequence of SEQ ID NO: 5; (b) CDR-H2comprising the amino acid sequence of SEQ ID NO: 6; and (c) CDR-H3comprising the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the anti-LILRB2 antibody comprises at least one, atleast two, or all three light chain CDR sequences selected from (a)CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8; (b) CDR-L2comprising the amino acid sequence of SEQ ID NO: 9; and (c) CDR-L3comprising the amino acid sequence of SEQ ID NO: 10.

In some embodiments, any of the six CDRs provided herein can be combinedas subparts with any of the other CDRs provided herein, for a total ofsix CDRs in a construct. Thus, in some embodiments, two CDRs from afirst antibody (for example, CDR-H1 and CDR-H2) can be combined withfour CDRs from a second antibody (CDR-H3, CDR-L1, CDR-L2, and CDR-L3).In some embodiments, two or fewer residues in one or more of the CDRscan be replaced to obtain a variant thereof. In some embodiments, two orfewer residues can be replaced in 1, 2, 3, 4, 5, or 6 of the CDRs.

In particular embodiments, an anti-LILRB2 antibody comprises a variableheavy chain (V_(H)) domain sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO: 3. In some embodiments, a V_(H) sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity contains substitutions (for example, conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-LILRB2 antibody comprising that sequence retainsthe ability to bind to LILRB2. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, and/or deleted in SEQ IDNO: 3. In some embodiments, substitutions, insertions, or deletionsoccur in regions outside the CDRs (that is, in the FRs). Optionally, theanti-LILRB2 antibody comprises the V_(H) sequence of SEQ ID NO: 3,including post-translational modifications of that sequence.

In some embodiments, the V_(H) comprises: (a) CDR-H1 comprising theamino acid sequence of SEQ ID NO: 5; (b) CDR-H2 comprising the aminoacid sequence of SEQ ID NO: 6; and (c) CDR-H3 comprising the amino acidsequence of SEQ ID NO: 7.

In some embodiments, an anti-LILRB2 antibody is provided, wherein theantibody comprises a variable light chain (V_(L)) domain having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 4. In someembodiments, a V_(L) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity contains substitutions (for example,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-LILRB2 antibody comprising that sequenceretains the ability to bind to LILRB2. In some embodiments, a total of 1to 10 amino acids have been substituted, inserted, and/or deleted in SEQID NO: 4. In some embodiments, the substitutions, insertions, ordeletions occur in regions outside the CDRs (that is, in the FRs).Optionally, the anti-LILRB2 antibody comprises the V_(L) sequence of SEQID NO: 4, including post-translational modifications of that sequence.

In some embodiments, the V_(L) comprises: (a) CDR-L1 comprising theamino acid sequence of SEQ ID NO: 8; (b) CDR-L2 comprising the aminoacid sequence of SEQ ID NO: 9; and (c) CDR-L3 comprising the amino acidsequence of SEQ ID NO: 10.

In some embodiments, an anti-LILRB2 antibody comprises a V_(H) domainsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:3 and a V_(L) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ IDNO: 4. In some embodiments, a V_(H) domain sequence having at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (for example, conservative substitutions), insertions, ordeletions relative to the reference sequence, and a V_(L) domainsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity contains substitutions (for example, conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-LILRB2 antibody comprising that sequence retainsthe ability to bind to LILRB2. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, and/or deleted in SEQ IDNO: 3. In some embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted, and/or deleted in SEQ ID NO: 4. In someembodiments, substitutions, insertions, or deletions occur in regionsoutside the CDRs (that is, in the FRs). Optionally, the anti-LILRB2antibody comprises the V_(H) domain sequence in SEQ ID NO: 3 and theV_(L) domain sequence of SEQ ID NO: 4, including post-translationalmodifications of one or both sequence.

In some embodiments, an anti-LILRB2 antibody comprises a V_(H) domain asin any of the embodiments provided herein, and a V_(L) domain as in anyof the embodiments provided herein. In some embodiments, the antibodycomprises the V_(H) and V_(L) domain sequences of SEQ ID NO: 3 and SEQID NO: 4, respectively, including post-translational modifications ofthose sequences.

In some embodiments, an anti-LILRB2 antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 1 and a light chaincomprising the amino acid sequence of SEQ ID NO: 2. In some aspects, ananti-LILRB2 antibody comprises at least one, two, three, four, five, orsix CDRs selected from (a) CDR-H1 comprising the amino acid sequence ofSEQ ID NO: 25; (b) CDR-H2 comprising the amino acid sequence of SEQ IDNO: 26; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27;(d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (e)CDR-L2 comprising the amino acid sequence of SEQ ID NO: 29; and (f)CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30.

In some embodiments, an anti-LILRB2 antibody is provided that competeswith an anti-LILRB2 antibody described herein. In some embodiments, anantibody that competes for binding (i.e., cross-competes) with any ofthe antibodies provided herein can be made and/or used.

In some embodiments, the anti-LILRB2 antibody comprises at least one, atleast two, or all three heavy chain CDR sequences selected from (a)CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25; (b) CDR-H2comprising the amino acid sequence of SEQ ID NO: 26; and (c) CDR-H3comprising the amino acid sequence of SEQ ID NO: 27.

In some embodiments, the anti-LILRB2 antibody comprises at least one, atleast two, or all three light chain CDR sequences selected from (a)CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28; (b) CDR-L2comprising the amino acid sequence of SEQ ID NO: 29; and (c) CDR-L3comprising the amino acid sequence of SEQ ID NO: 30.

In some embodiments, any of the six CDRs provided herein can be combinedas subparts with any of the other CDRs provided herein, for a total ofsix CDRs in a construct. Thus, in some embodiments, two CDRs from afirst antibody (for example, CDR-H1 and CDR-H2) can be combined withfour CDRs from a second antibody (CDR-H3, CDR-L1, CDR-L2, and CDR-L3).In some embodiments, two or fewer residues in one or more of the CDRscan be replaced to obtain a variant thereof. In some embodiments, two orfewer residues can be replaced in 1, 2, 3, 4, 5, or 6 of the CDRs.

In particular embodiments, an anti-LILRB2 antibody comprises a variableheavy chain (V_(H)) domain sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO: 23. In some embodiments, a V_(H) sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity contains substitutions (for example, conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-LILRB2 antibody comprising that sequence retainsthe ability to bind to LILRB2. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, and/or deleted in SEQ IDNO: 23. In some embodiments, substitutions, insertions, or deletionsoccur in regions outside the CDRs (that is, in the FRs). Optionally, theanti-LILRB2 antibody comprises the V_(H) sequence of SEQ ID NO: 23,including post-translational modifications of that sequence.

In some embodiments, the V_(H) comprises: (a) CDR-H1 comprising theamino acid sequence of SEQ ID NO: 25; (b) CDR-H2 comprising the aminoacid sequence of SEQ ID NO: 26; and (c) CDR-H3 comprising the amino acidsequence of SEQ ID NO: 27.

In some embodiments, an anti-LILRB2 antibody is provided, wherein theantibody comprises a variable light chain (V_(L)) domain having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 24. In someembodiments, a V_(L) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity contains substitutions (for example,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-LILRB2 antibody comprising that sequenceretains the ability to bind to LILRB2. In some embodiments, a total of 1to 10 amino acids have been substituted, inserted, and/or deleted in SEQID NO: 24. In some embodiments, the substitutions, insertions, ordeletions occur in regions outside the CDRs (that is, in the FRs).Optionally, the anti-LILRB2 antibody comprises the V_(L) sequence of SEQID NO: 24, including post-translational modifications of that sequence.

In some embodiments, the V_(L) comprises: (a) CDR-L1 comprising theamino acid sequence of SEQ ID NO: 28; (b) CDR-L2 comprising the aminoacid sequence of SEQ ID NO: 29; and (c) CDR-L3 comprising the amino acidsequence of SEQ ID NO: 30.

In some embodiments, an anti-LILRB2 antibody comprises a V_(H) domainsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:23 and a V_(L) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ IDNO: 24. In some embodiments, a V_(H) domain sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (for example, conservative substitutions), insertions, ordeletions relative to the reference sequence, and a V_(L) domainsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity contains substitutions (for example, conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-LILRB2 antibody comprising that sequence retainsthe ability to bind to LILRB2. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, and/or deleted in SEQ IDNO: 23. In some embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted, and/or deleted in SEQ ID NO: 24. In someembodiments, substitutions, insertions, or deletions occur in regionsoutside the CDRs (that is, in the FRs). Optionally, the anti-LILRB2antibody comprises the V_(H) domain sequence in SEQ ID NO: 23 and theV_(L) domain sequence of SEQ ID NO: 24, including post-translationalmodifications of one or both sequence.

In some embodiments, an anti-LILRB2 antibody comprises a V_(H) domain asin any of the embodiments provided herein, and a V_(L) domain as in anyof the embodiments provided herein. In some embodiments, the antibodycomprises the V_(H) and V_(L) domain sequences of SEQ ID NO: 23 and SEQID NO: 24, respectively, including post-translational modifications ofthose sequences.

In some embodiments, an anti-LILRB2 antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 21 and a light chaincomprising the amino acid sequence of SEQ ID NO: 22.

In some aspects, an anti-LILRB2 antibody comprises at least one, two,three, four, five, or six CDRs selected from (a) CDR-H1 comprising theamino acid sequence of SEQ ID NO: 35; (b) CDR-H2 comprising the aminoacid sequence of SEQ ID NO: 36; (c) CDR-H3 comprising the amino acidsequence of SEQ ID NO: 37; (d) CDR-L1 comprising the amino acid sequenceof SEQ ID NO: 38; (e) CDR-L2 comprising the amino acid sequence of SEQID NO: 39; and (f) CDR-L3 comprising the amino acid sequence of SEQ IDNO: 40.

In some embodiments, an anti-LILRB2 antibody is provided that competeswith an anti-LILRB2 antibody described herein. In some embodiments, anantibody that competes for binding (i.e., cross-competes) with any ofthe antibodies provided herein can be made and/or used.

In some embodiments, the anti-LILRB2 antibody comprises at least one, atleast two, or all three heavy chain CDR sequences selected from (a)CDR-H1 comprising the amino acid sequence of SEQ ID NO: 35; (b) CDR-H2comprising the amino acid sequence of SEQ ID NO: 36; and (c) CDR-H3comprising the amino acid sequence of SEQ ID NO: 37.

In some embodiments, the anti-LILRB2 antibody comprises at least one, atleast two, or all three light chain CDR sequences selected from (a)CDR-L1 comprising the amino acid sequence of SEQ ID NO: 38; (b) CDR-L2comprising the amino acid sequence of SEQ ID NO: 39; and (c) CDR-L3comprising the amino acid sequence of SEQ ID NO: 40.

In some embodiments, any of the six CDRs provided herein can be combinedas subparts with any of the other CDRs provided herein, for a total ofsix CDRs in a construct. Thus, in some embodiments, two CDRs from afirst antibody (for example, CDR-H1 and CDR-H2) can be combined withfour CDRs from a second antibody (CDR-H3, CDR-L1, CDR-L2, and CDR-L3).In some embodiments, two or fewer residues in one or more of the CDRscan be replaced to obtain a variant thereof. In some embodiments, two orfewer residues can be replaced in 1, 2, 3, 4, 5, or 6 of the CDRs.

In particular embodiments, an anti-LILRB2 antibody comprises a variableheavy chain (V_(H)) domain sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO: 33. In some embodiments, a V_(H) sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity contains substitutions (for example, conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-LILRB2 antibody comprising that sequence retainsthe ability to bind to LILRB2. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, and/or deleted in SEQ IDNO: 33. In some embodiments, substitutions, insertions, or deletionsoccur in regions outside the CDRs (that is, in the FRs). Optionally, theanti-LILRB2 antibody comprises the V_(H) sequence of SEQ ID NO: 33,including post-translational modifications of that sequence.

In some embodiments, the V_(H) comprises: (a) CDR-H1 comprising theamino acid sequence of SEQ ID NO: 35; (b) CDR-H2 comprising the aminoacid sequence of SEQ ID NO: 36; and (c) CDR-H3 comprising the amino acidsequence of SEQ ID NO: 37.

In some embodiments, an anti-LILRB2 antibody is provided, wherein theantibody comprises a variable light chain (V_(L)) domain having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 34. In someembodiments, a V_(L) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity contains substitutions (for example,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-LILRB2 antibody comprising that sequenceretains the ability to bind to LILRB2. In some embodiments, a total of 1to 10 amino acids have been substituted, inserted, and/or deleted in SEQID NO: 34. In some embodiments, the substitutions, insertions, ordeletions occur in regions outside the CDRs (that is, in the FRs).Optionally, the anti-LILRB2 antibody comprises the V_(L) sequence of SEQID NO: 34, including post-translational modifications of that sequence.

In some embodiments, the V_(L) comprises: (a) CDR-L1 comprising theamino acid sequence of SEQ ID NO: 38; (b) CDR-L2 comprising the aminoacid sequence of SEQ ID NO: 39; and (c) CDR-L3 comprising the amino acidsequence of SEQ ID NO: 40.

In some embodiments, an anti-LILRB2 antibody comprises a V_(H) domainsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:33 and a V_(L) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ IDNO: 34. In some embodiments, a V_(H) domain sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (for example, conservative substitutions), insertions, ordeletions relative to the reference sequence, and a V_(L) domainsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity contains substitutions (for example, conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-LILRB2 antibody comprising that sequence retainsthe ability to bind to LILRB2. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, and/or deleted in SEQ IDNO: 33. In some embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted, and/or deleted in SEQ ID NO: 34. In someembodiments, substitutions, insertions, or deletions occur in regionsoutside the CDRs (that is, in the FRs). Optionally, the anti-LILRB2antibody comprises the V_(H) domain sequence in SEQ ID NO: 33 and theV_(L) domain sequence of SEQ ID NO: 34, including post-translationalmodifications of one or both sequence.

In some embodiments, an anti-LILRB2 antibody comprises a V_(H) domain asin any of the embodiments provided herein, and a V_(L) domain as in anyof the embodiments provided herein. In some embodiments, the antibodycomprises the V_(H) and V_(L) domain sequences of SEQ ID NO: 33 and SEQID NO: 34, respectively, including post-translational modifications ofthose sequences.

In some embodiments, an anti-LILRB2 antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 31 and a light chaincomprising the amino acid sequence of SEQ ID NO: 32.

In some aspects, an anti-LILRB2 antibody comprises at least one, two,three, four, five, or six CDRs selected from (a) CDR-H1 comprising theamino acid sequence of SEQ ID NO: 45; (b) CDR-H2 comprising the aminoacid sequence of SEQ ID NO: 46; (c) CDR-H3 comprising the amino acidsequence of SEQ ID NO: 47; (d) CDR-L1 comprising the amino acid sequenceof SEQ ID NO: 48; (e) CDR-L2 comprising the amino acid sequence of SEQID NO: 49; and (f) CDR-L3 comprising the amino acid sequence of SEQ IDNO: 50.

In some embodiments, an anti-LILRB2 antibody is provided that competeswith an anti-LILRB2 antibody described herein. In some embodiments, anantibody that competes for binding (i.e., cross-competes) with any ofthe antibodies provided herein can be made and/or used.

In some embodiments, the anti-LILRB2 antibody comprises at least one, atleast two, or all three heavy chain CDR sequences selected from (a)CDR-H1 comprising the amino acid sequence of SEQ ID NO: 45; (b) CDR-H2comprising the amino acid sequence of SEQ ID NO: 46; and (c) CDR-H3comprising the amino acid sequence of SEQ ID NO: 47.

In some embodiments, the anti-LILRB2 antibody comprises at least one, atleast two, or all three light chain CDR sequences selected from (a)CDR-L1 comprising the amino acid sequence of SEQ ID NO: 48; (b) CDR-L2comprising the amino acid sequence of SEQ ID NO: 49; and (c) CDR-L3comprising the amino acid sequence of SEQ ID NO: 50.

In some embodiments, any of the six CDRs provided herein can be combinedas subparts with any of the other CDRs provided herein, for a total ofsix CDRs in a construct. Thus, in some embodiments, two CDRs from afirst antibody (for example, CDR-H1 and CDR-H2) can be combined withfour CDRs from a second antibody (CDR-H3, CDR-L1, CDR-L2, and CDR-L3).In some embodiments, two or fewer residues in one or more of the CDRscan be replaced to obtain a variant thereof. In some embodiments, two orfewer residues can be replaced in 1, 2, 3, 4, 5, or 6 of the CDRs.

In particular embodiments, an anti-LILRB2 antibody comprises a variableheavy chain (V_(H)) domain sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO: 43. In some embodiments, a V_(H) sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity contains substitutions (for example, conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-LILRB2 antibody comprising that sequence retainsthe ability to bind to LILRB2. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, and/or deleted in SEQ IDNO: 43. In some embodiments, substitutions, insertions, or deletionsoccur in regions outside the CDRs (that is, in the FRs). Optionally, theanti-LILRB2 antibody comprises the V_(H) sequence of SEQ ID NO: 43,including post-translational modifications of that sequence.

In some embodiments, the V_(H) comprises: (a) CDR-H1 comprising theamino acid sequence of SEQ ID NO: 45; (b) CDR-H2 comprising the aminoacid sequence of SEQ ID NO: 46; and (c) CDR-H3 comprising the amino acidsequence of SEQ ID NO: 47.

In some embodiments, an anti-LILRB2 antibody is provided, wherein theantibody comprises a variable light chain (V_(L)) domain having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 44. In someembodiments, a V_(L) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity contains substitutions (for example,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-LILRB2 antibody comprising that sequenceretains the ability to bind to LILRB2. In some embodiments, a total of 1to 10 amino acids have been substituted, inserted, and/or deleted in SEQID NO: 44. In some embodiments, the substitutions, insertions, ordeletions occur in regions outside the CDRs (that is, in the FRs).Optionally, the anti-LILRB2 antibody comprises the V_(L) sequence of SEQID NO: 44, including post-translational modifications of that sequence.

In some embodiments, the V_(L) comprises: (a) CDR-L1 comprising theamino acid sequence of SEQ ID NO: 48; (b) CDR-L2 comprising the aminoacid sequence of SEQ ID NO: 49; and (c) CDR-L3 comprising the amino acidsequence of SEQ ID NO: 50.

In some embodiments, an anti-LILRB2 antibody comprises a V_(H) domainsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:43 and a V_(L) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ IDNO: 44. In some embodiments, a V_(H) domain sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (for example, conservative substitutions), insertions, ordeletions relative to the reference sequence, and a V_(L) domainsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity contains substitutions (for example, conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-LILRB2 antibody comprising that sequence retainsthe ability to bind to LILRB2. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, and/or deleted in SEQ IDNO: 43. In some embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted, and/or deleted in SEQ ID NO: 44. In someembodiments, substitutions, insertions, or deletions occur in regionsoutside the CDRs (that is, in the FRs). Optionally, the anti-LILRB2antibody comprises the V_(H) domain sequence in SEQ ID NO: 43 and theV_(L) domain sequence of SEQ ID NO: 44, including post-translationalmodifications of one or both sequence.

In some embodiments, an anti-LILRB2 antibody comprises a V_(H) domain asin any of the embodiments provided herein, and a V_(L) domain as in anyof the embodiments provided herein. In some embodiments, the antibodycomprises the V_(H) and V_(L) domain sequences of SEQ ID NO: 43 and SEQID NO: 44, respectively, including post-translational modifications ofthose sequences.

In some embodiments, an anti-LILRB2 antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 41 and a light chaincomprising the amino acid sequence of SEQ ID NO: 42.

In some embodiments, the anti-LILRB2 antibody comprises the six CDRs asdescribed in any of the embodiments above and specifically binds toLILRB2 (e.g., human LILRB2). In some embodiments, the anti-LILRB2antibody comprises the six CDRs of any of the embodiments describedabove, specifically binds to LILRB2, and blocks binding of HLA-G and/orHLA-A2 to LILRB2 (e.g., human LILRB2). In some embodiments, theanti-LILRB2 antibody comprises the six CDRs as described in any of theembodiments above, specifically binds to LILRB2 (e.g., human LILRB2),and causes conversion of M2-like macrophages to M1-like macrophages.

In some embodiments, an anti-LILRB2 antibody comprises a variable heavychain region and a variable light chain region. In some embodiments, ananti-LILRB2 antibody comprises at least one heavy chain comprising avariable heavy chain region and at least a portion of a heavy chainconstant region, and at least one light chain comprising a variablelight chain region and at least a portion of a light chain constantregion. In some embodiments, an anti-LILRB2 antibody comprises two heavychains, wherein each heavy chain comprises a variable heavy chain regionand at least a portion of a constant heavy chain region, and two lightchains, wherein each light chain comprises a variable light chain regionand at least a portion of a constant light chain region. As used herein,a single-chain Fv (scFv), or any other antibody that comprises, forexample, a single polypeptide chain comprising all six CDRs (three heavychain CDRs and three light chain CDRs) is considered to have a heavychain and a light chain. In some embodiments, the heavy chain is theregion of the anti-LILRB2 antibody that comprises the three heavy chainCDRs. In some embodiments, the light chain is the region of theanti-LILRB2 antibody that comprises the three light chain CDRs.

In some embodiments, antibodies which compete with the anti-LILRB2antibodies provided herein for binding to LILRB2 are provided. In someembodiments, antibodies cross-compete with the anti-LILRB2 antibodiesprovided herein for binding to an epitope on LILRB2.

In some embodiments, competition assays may be used to identify amonoclonal antibody that competes with an anti-LILRB2 antibody describedherein for binding to LILRB2. Competition assays can be used todetermine whether two antibodies bind the same epitope by recognizingidentical or sterically overlapping epitopes or one antibodycompetitively inhibits binding of another antibody to the antigen. Insome embodiments, such a competing antibody binds to the same epitopethat is bound by an antibody described herein. Exemplary competitionassays include, but are not limited to, routine assays such as thoseprovided in Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Detailedexemplary methods for mapping an epitope to which an antibody binds areprovided in Morris (1996) “Epitope Mapping Protocols,” in Methods inMolecular Biology vol. 66 (Humana Press, Totowa, N.J.). In someembodiments, two antibodies are said to bind to the same epitope if eachblocks binding of the other by 50% or more. In some embodiments, theantibody that competes with an anti-LILRB2 antibody described herein isa chimeric, humanized or human antibody. In some embodiments, anantibody that competes with a chimeric, humanized, or human anti-LILRB2antibody as described herein is provided.

In some embodiments, antibodies that bind to any one or more of theepitopes of the antibodies provided herein are provided. In someembodiments, antibodies that bind and overlap an epitope to which thepresent antibodies bind to are provided. In some embodiments, anantibody is provided that competes with at least one of the antibodiesprovided herein. In some embodiments, an antibody is provided thatcompetes with at least two of the antibodies provided herein. In someembodiments, an antibody is provided that competes with at least threeof the antibodies provided herein. In some embodiments, the antibodybinds to an overlapping epitope as an antibody described in the examplesherein. In some embodiments, the entire epitope is bound and/orobstructed by the competing antibody. In some embodiments, a part of theepitope is bound and/or obstructed by the competing antibody. In someembodiments, the competing antibody's paratope binds to at least a partof the epitope of an antibody provided herein. In some embodiments, thecompeting antibody's paratope binds the target, and a different sectionof the competing antibody's structure obstruct at least a part of theepitope of an antibody provided herein.

Exemplary Chimeric Antibodies

In some embodiments, an antibody provided herein is a chimeric antibody.Certain chimeric antibodies are described, for example, in U.S. Pat. No.4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA,81:6851-6855. In one example, a chimeric antibody comprises a non-humanvariable region (for example, a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

Nonlimiting exemplary chimeric antibodies include chimeric antibodiescomprising the heavy and/or light chain variable regions of any of theanti-LILRB2 antibodies described herein. Nonlimiting exemplary chimericantibodies include chimeric antibodies comprising a CDR-H1, CDR-H2, andCDR-H3, and/or a CDR-L1, CDR-L2, and CDR-L3 of any of the anti-LILRB2antibodies described herein. In some embodiments, the chimericanti-LILRB2 antibody comprises the variable regions described above andbinds to LILRB2. In some embodiments, the chimeric anti-LILRB2 antibodycomprises the variable regions described above, binds to LILRB2, andcauses conversion of M2-like macrophages to M1-like macrophages.

In some embodiments, a chimeric anti-LILRB2 antibody comprises a heavychain comprising a variable region sequence that is at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to asequence selected from SEQ ID NOs: 3, 13, 23, 33, 43, 53, 63, 73, 83,93, 103, and 113 wherein the antibody binds LILRB2. In some embodiments,a chimeric anti-LILRB2 antibody comprises a light chain comprising avariable region sequence that is at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to a sequence selected fromSEQ ID NOs: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, and 114 whereinthe antibody binds LILRB2. In some embodiments, a chimeric anti-LILRB2antibody comprises a heavy chain comprising a variable region sequencethat is at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identical to a sequence selected from SEQ ID NOs: 3, 13, 23, 33, 43,53, 63, 73, 83, 93, 103, and 113; and a light chain comprising avariable region sequence that is at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to a sequence selected fromSEQ ID NOs: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, and 114; whereinthe antibody binds LILRB2.

Exemplary chimeric anti-LILRB2 antibodies also include chimericantibodies that compete for binding to LILRB2 with an antibody orfragment thereof described herein. Thus, in some embodiments, a chimericanti-LILRB2 antibody is provided that competes for binding to LILRB2with any of the LILRB2 antibodies described herein, or fragment thereof.In some embodiments, the antibody competes for binding to LILRB2 andcauses conversion of M2-like macrophages to M1-like macrophages.

In some embodiments, a chimeric antibody described herein comprises oneor more human constant regions. In some embodiments, the human heavychain constant region is of an isotype selected from IgA, IgG, and IgD.In some embodiments, the human light chain constant region is of anisotype selected from K and A. In some embodiments, a chimeric antibodydescribed herein comprises a human IgG constant region. In someembodiments, a chimeric antibody described herein comprises a human IgG4heavy chain constant region. In some embodiments, a chimeric antibodydescribed herein comprises a human IgG4 constant region and a human Klight chain.

As noted above, whether or not effector function is desirable may dependon the particular method of treatment intended for an antibody. Thus, insome embodiments, when effector function is desirable, a chimericanti-LILRB2 antibody comprising a human IgG1 heavy chain constant regionor a human IgG3 heavy chain constant region is selected. In someembodiments, when effector function is not desirable, a chimericanti-LILRB2 antibody comprising a human IgG4 or IgG2 heavy chainconstant region is selected.

Exemplary Humanized Antibodies

In some embodiments, humanized antibodies that bind LILRB2 are provided.Humanized antibodies are useful as therapeutic molecules becausehumanized antibodies reduce or eliminate the human immune response ascompared to non-human antibodies, which can result in an immune responseto an antibody therapeutic (such as the human anti-mouse antibody (HAMA)response), and decreased effectiveness of the therapeutic.

In some embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which CDRs, (or portions thereof) are derivedfrom a non-human antibody, and FRs (or portions thereof) are derivedfrom human antibody sequences. A humanized antibody optionally will alsocomprise at least a portion of a human constant region. In someembodiments, some FR residues in a humanized antibody are substitutedwith corresponding residues from a non-human antibody (for example, theantibody from which the CDR residues are derived), for example, torestore or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, forexample, in Almagro and Fransson, 2008, Front. Biosci., 13: 1619-1633,and are further described, for example, in Riechmann et al., 1988,Nature, 332:323-329; Queen et al., 1989, Proc. Natl Acad. Sci. USA, 86:10029-10033; U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and7,087,409; Kashmiri et al., 2005, Methods, 36:25-34; Padlan, 1991, Mol.Immunol., 28:489-498 (describing “resurfacing”); Dall'Acqua et al.,2005, Methods, 36:43-60 (describing “FR shuffling”); and Osbourn et al.,2005, Methods, 36:61-68 and Klimka et al., 2000, Br. J. Cancer,83:252-260 (describing the “guided selection” approach to FR shuffling).

Human framework regions that can be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, for example, Sims et al., 1993, J. Immunol. 151:2296);framework regions derived from the consensus sequence of humanantibodies of a particular subgroup of light or heavy chain variableregions (see, for example, Carter et al., 1992, Proc. Natl. Acad. Sci.USA, 89:4285; and Presta et al., 1993, J. Immunol., 151:2623); humanmature (somatically mutated) framework regions or human germlineframework regions (see, for example, Almagro and Fransson, 2008, Front.Biosci., 13:1619-1633); and framework regions derived from screening FRlibraries (see, for example, Baca et al., 1997, J. Biol. Chem., 272:10678-10684 and Rosok et al., 1996, J. Biol. Chem., 271:22611-22618).

Nonlimiting exemplary humanized antibodies include antibodies comprisinga V_(H) domain selected from SEQ ID NOs: 53, 63, 73, 83, 93, 103, and113 and/or a V_(L) domain selected from SEQ ID NOs: 54, 64, 74, 84, 94,104, and 114, or any one, two, three, four, five, or six CDRs thereof.In some embodiments, the humanized anti-LILRB2 antibody comprises theCDRs described above and binds to LILRB2. In some embodiments, thehumanized anti-LILRB2 antibody comprises the CDRs described above, bindsto LILRB2 and causes conversion of M2-like macrophages to M1-likemacrophages.

In some embodiments, a humanized anti-LILRB2 antibody comprises a heavychain comprising a variable region sequence that is at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to asequence selected from SEQ ID NOs: 53, 63, 73, 83, 93, 103, and 113 andwherein the antibody binds LILRB2. In some embodiments, a humanizedanti-LILRB2 antibody comprises a light chain comprising a variableregion sequence that is at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to a sequence selected from SEQ IDNOs: 54, 64, 74, 84, 94, 104, and 114, wherein the antibody bindsLILRB2. In some embodiments, a humanized anti-LILRB2 antibody comprisesa heavy chain comprising a variable region sequence that is at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% identicalto a sequence selected from SEQ ID NOs: 53, 63, 73, 83, 93, and 113 anda light chain comprising a variable region sequence that is at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% identicalto a sequence selected from SEQ ID NOs: 54, 64, 74, 84, 94, 104, and 114wherein the antibody binds LILRB2.

In some embodiments, any one or more of the CDR sequences providedherein are maintained, while the remain heavy and/or light chain region(that is, FR1, FR2, FR3, and FR4) is at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identical to a sequenceselected from SEQ ID NOs: 53, 54, 63, 64, 73, 74, 83, 84, 93, 94, 103,104, 113, and 114.

In some embodiments, a humanized anti-LILRB2 antibody comprises at leastone of the CDRs discussed herein. That is, in some embodiments, ahumanized anti-LILRB2 antibody comprises at least one CDR selected froma CDR-H1 discussed herein, a CDR-H2 discussed herein, a CDR-H3 discussedherein, a CDR-L1 discussed herein, a CDR-L2 discussed herein, and aCDR-L3 discussed herein. Further, in some embodiments, a humanizedanti-LILRB2 antibody comprises at least one mutated CDR based on a CDRdiscussed herein, wherein the mutated CDR comprises 1, 2, 3, or 4 aminoacid substitutions relative to the CDR discussed herein. In someembodiments, one or more of the amino acid substitutions areconservative amino acid substitutions. One skilled in the art can selectone or more suitable conservative amino acid substitutions for aparticular CDR sequence, wherein the suitable conservative amino acidsubstitutions are not predicted to significantly alter the bindingproperties of the antibody comprising the mutated CDR.

Exemplary humanized anti-LILRB2 antibodies also include antibodies thatcompete for binding to LILRB2 with an antibody or fragment thereofdescribed herein. In some embodiments, a humanized anti-LILRB2 antibodyis provided that competes for binding to LILRB2 with any anti-LILRB2antibody described herein, or fragment thereof, and causes conversion ofM2-like macrophages to M1-like macrophages.

Exemplary Human Antibodies

In some embodiments, an anti-LILRB2 antibody provided herein is a humanantibody. Human antibodies can be produced using various techniquesknown in the art. Human antibodies are described generally in van Dijkand van de Winkel, 2001, Curr. Opin. Pharmacol., 5:368-374 and Lonberg,2008, Curr. Opin. Immunol., 20:450-459. In some embodiments, the humanantibody is not a naturally occurring antibody. In some embodiments, thehuman antibody is a monoclonal antibody; thus, in some embodiments, eachof the human antibodies in a set can bind to the same epitope on theantigen.

Human antibodies can be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, 2005, Nat. Biotech., 23: 1117-1125. Seealso, for example, U.S. Pat. Nos. 6,075,181 and 6,150,584 describingXENOMOUSE™ technology; U.S. Pat. No. 5,770,429 describing HUMAB®technology; U.S. Pat. No. 7,041,870 describing K-M MOUSE® technology,and U.S. Patent Application Publication No. US 2007/0061900, describingVELOCIMOUSE® technology). Human variable regions from intact antibodiesgenerated by such animals may be further modified, for example, bycombining with a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. See, for example,Kozbor, 1984, J. Immunol., 133: 3001; Brodeur et al., MonoclonalAntibody Production Techniques and Applications, pp. 51-63 (MarcelDekker, Inc., New York, 1987); and Boerner et al., 1991, J. Immunol.,147:86). Human antibodies generated via human B-cell hybridomatechnology are also described in Li et al., 2006, Proc. Natl. Acad. Sci.USA, 103:3557-3562. Additional methods include those described, forexample, in U.S. Pat. No. 7,189,826 (describing production of monoclonalhuman IgM antibodies from hybridoma cell lines) and Ni, 2006, XiandaiMianyixue, 26(4):265-268 (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, 2005, Histology and Histopathology, 20(3):927-937 andVollmers and Brandlein, 2005, Methods and Findings in Experimental andClinical Pharmacology, 27(3): 185-191.

Human antibodies can also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

Antibodies may be isolated by screening combinatorial libraries forantibodies with the desired activity or activities. For example, avariety of methods are known in the art for generating phage displaylibraries and screening such libraries for antibodies possessing thedesired binding characteristics. Such methods are reviewed, for example,in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brienet al., ed., Human Press, Totowa, N.J., 2001) and further described, forexample, in the McCafferty et al., 1990, Nature 348:552-554; Clackson etal., 1991, Nature, 352: 624-628; Marks et al., 1992, J. Mol. Biol., 222:581-597; Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, N.J., 2003); Sidhu et al., 2004J. Mol. Biol., 338(2): 299-310; Lee et al., 2004, J. Mol. Biol. 340(5):1073-1093; Fellouse, 2004, Proc. Natl. Acad. Sci. USA, 101(34):12467-12472; and Lee et al., 2004, J. Immunol. Methods, 284(1-2):119-132 and PCT publication WO 99/10494.

In certain phage display methods, repertoires of V_(H) and V_(L) genesare separately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., 1994, Ann. Rev.Immunol., 12:433-455. Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (for example, from human) to provide asingle source of antibodies to a wide range of non-self and alsoself-antigens without any immunization as described by Griffiths et al.,1993, EMBO J, 12:725-734. Finally, naive libraries can also be madesynthetically by cloning unrearranged V-gene segments from stem cells,and using PCR primers containing random sequence to encode the highlyvariable CDR3 regions and to accomplish rearrangement in vitro, asdescribed by Hoogenboom and Winter 1992, J. Mol. Biol., 227:381-388.Patent publications describing human antibody phage libraries include,for example: U.S. Pat. No. 5,750,373, and U.S. Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

In some embodiments, a chimeric human anti-LILRB2 antibody is provided,where the antibody comprises the variable region from a human antibodythat binds LILRB2 and the constant region from a different humanantibody. In some embodiments, a chimeric human anti-LILRB2 antibody,where the antibody comprises the CDRs from a human antibody that bindsLILRB2 and a framework from a different human antibody is provided. Insome embodiments, the antibody is not a naturally occurring humanantibody.

In some embodiments, a human anti-LILRB2 antibody comprises one or morehuman constant regions. In some embodiments, the human heavy chainconstant region is of an isotype selected from IgA, IgG, and IgD. Insome embodiments, the human light chain constant region is of an isotypeselected from K and A. In some embodiments, a human antibody describedherein comprises a human IgG constant region. In some embodiments, ahuman antibody described herein comprises a human IgG4 heavy chainconstant region. In some embodiments, a human antibody described hereincomprises a human IgG4 constant region and a human K light chain.

In some embodiments, when effector function is desirable, a humananti-LILRB2 antibody comprising a human IgG1 heavy chain constant regionor a human IgG3 heavy chain constant region is selected. In someembodiments, when effector function is not desirable, a humananti-LILRB2 antibody comprising a human IgG4 or IgG2 heavy chainconstant region is selected.

As noted herein, the term “human antibody” denotes the genus of possiblesequences for the antibody construct, rather than a source of theantibody.

Exemplary Antibody Constant Regions

In some embodiments, an antibody described herein comprises one or morehuman constant regions. In some embodiments, the human heavy chainconstant region is of an isotype selected from IgA, IgG, and IgD. Insome embodiments, the human light chain constant region is of an isotypeselected from K and A. In some embodiments, an antibody described hereincomprises a human IgG constant region. In some embodiments, an antibodydescribed herein comprises a human IgG4 heavy chain constant region. Insome embodiments, an antibody described herein comprises a human IgG4constant region and a human K light chain.

Throughout the present specification and claims unless explicitly statedor known to one skilled in the art, the numbering of the residues in animmunoglobulin heavy chain is that of the EU index as in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991), expresslyincorporated herein by reference. The “EU index as in Kabat” refers tothe residue numbering of the human IgG1 EU antibody.

As noted above, whether or not effector function is desirable may dependon the particular method of treatment intended for an antibody. Thus, insome embodiments, when effector function is desirable, an anti-LILRB2antibody comprising a human IgG1 heavy chain constant region or a humanIgG3 heavy chain constant region is selected. In some embodiments, wheneffector function is not desirable, an anti-LILRB2 antibody comprising ahuman IgG4 or IgG2 heavy chain constant region is selected.

In some embodiments, an antibody comprises a variant Fc region has atleast one amino acid substitution compared to the Fc region of awild-type IgG or a wild-type antibody. In some embodiments, the variantFc region has two or more amino acid substitutions in the Fc region ofthe wild-type antibody. In some embodiments, the variant Fc region hasthree or more amino acid substitutions in the Fc region of the wild-typeantibody. In some embodiments, the variant Fc region has at least one,two or three or more Fc region amino acid substitutions describedherein. In some embodiments, the variant Fc region herein will possessat least about 80% homology with a native sequence Fc region and/or withan Fc region of a parent polypeptide. In some embodiments, the variantFc region herein will possess at least about 90% homology with a nativesequence Fc region and/or with an Fc region of a parent polypeptide. Insome embodiments, the variant Fc region herein will possess at leastabout 95% homology with a native sequence Fc region and/or with an Fcregion of a parent polypeptide.

In some embodiments, an antibody provided herein is altered to increaseor decrease the extent to which the antibody is glycosylated. Additionor deletion of glycosylation sites to an antibody may be convenientlyaccomplished by altering the amino acid sequence such that one or moreglycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, for example, Wright et al., 1997, TIBTECH, 15:26-32. Theoligosaccharide may include various carbohydrates, for example, mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody may be made in order to create antibodyvariants with certain improved properties.

In some embodiments, antibody variants are provided having acarbohydrate structure that lacks fucose attached (directly orindirectly) to an Fc region. For example, the amount of fucose in suchantibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from20% to 40%. The amount of fucose is determined by calculating theaverage amount of fucose within the sugar chain at Asn297, relative tothe sum of all glycostructures attached to Asn 297 (for example,complex, hybrid and high mannose structures) as measured by MALDI-TOFmass spectrometry, as described in WO 2008/077546, for example. Asn297refers to the asparagine residue located at about position 297 in the Fcregion (EU numbering of Fc region residues); however, Asn297 may also belocated about ±3 amino acids upstream or downstream of position 297,that is, between positions 294 and 300, due to minor sequence variationsin antibodies. Such fucosylation variants may have improved ADCCfunction. See, for example, U.S. Patent Publication Nos. U.S.2003/0157108 (Presta, L.); U.S. 2004/0093621 (Kyowa Hakko Kogyo Co.,Ltd). Examples of publications related to “defucosylated” or“fucose-deficient” antibody variants include: U.S. 2003/0157108; WO2000/61739; WO 2001/29246; U.S. 2003/0115614; U.S. 2002/0164328; U.S.2004/0093621; U.S. 2004/0132140; U.S. 2004/0110704; U.S. 2004/0110282;U.S. 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol.336:1239-1249 (2004); Yamane-Ohnuki et al., 2004, Biotech. Bioeng. 87:614. Examples of cell lines capable of producing defucosylatedantibodies include Lec13 CHO cells deficient in protein fucosylation(Ripka et al., 1986, Arch. Biochem. Biophys. 249: 533-545; U.S. PatentApplication No. U.S. 2003/0157108 A1 (Presta, L); and WO 2004/056312 A1,(Adams et al., especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, forexample, Yamane-Ohnuki et al., 2004, Biotech. Bioeng. 87: 614; Kanda, Y.et al., 2006, Biotechnol. Bioeng., 94(4):680-688; and WO 2003/085107).

Antibody variants are further provided with bisected oligosaccharides,for example, in which a biantennary oligosaccharide attached to the Fcregion of the antibody is bisected by GlcNAc. Such antibody variants mayhave reduced fucosylation and/or improved ADCC function. Examples ofsuch antibody variants are described, for example, in WO 2003/011878(Jean-Mairet et al.); U.S. Patent No. U.S. Pat. No. 6,602,684 (Umana etal.); and U.S. 2005/0123546 (Umana et al.). Antibody variants with atleast one galactose residue in the oligosaccharide attached to the Fcregion are also provided. Such antibody variants may have improved CDCfunction. Such antibody variants are described, for example, in WO1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764(Raju, S.).

Antibody variants are also provided with amino-terminal leaderextensions. For example, one or more amino acid residues of theamino-terminal leader sequence are present at the amino-terminus of anyone or more heavy or light chains of an antibody. An exemplaryamino-terminal leader extension comprises or consists of three aminoacid residues, VHS, present on one or both light chains of an antibodyvariant.

The in vivo or serum half-life of human FcRn high affinity bindingpolypeptides can be assayed, for example, in transgenic mice, in humans,or in non-human primates to which the polypeptides with a variant Fcregion are administered. See also, for example, Petkova et al., 2006,International Immunology 18(12):1759-1769.

In some embodiments, the antibody variant mediates ADCC in the presenceof human effector cells more effectively than a parent antibody. In someembodiments, the antibody variant is substantially more effective atmediating ADCC in vitro, when the amounts of polypeptide variant andparent antibody used in the assay are essentially the same. In someembodiments, the antibody variant is substantially more effective atmediating ADCC in vivo, when the amounts of polypeptide variant andparent antibody used in the assay are essentially the same. Generally,such variants will be identified using the in vitro ADCC assay as hereindisclosed, but other assays or methods for determining ADCC activity,for example in an animal model etc., are contemplated.

Exemplary Antibody Conjugates

In some embodiments, an anti-LILRB2 antibody is conjugated to anothermolecule. In some embodiments, the additional molecule can be adetectable marker, such as a label. In some embodiments, the additionalmolecule can be a therapeutic molecule, such as a cytotoxic agent. Insome embodiments, a label and/or a cytotoxic agent can be conjugated tothe antibody. As used herein, a label is a moiety that facilitatesdetection of the antibody and/or facilitates detection of a molecule towhich the antibody binds. Nonlimiting exemplary labels include, but arenot limited to, radioisotopes, fluorescent groups, enzymatic groups,chemiluminescent groups, biotin, epitope tags, metal-binding tags, etc.One skilled in the art can select a suitable label according to thespecific application.

As used herein, a cytotoxic agent is a moiety that reduces theproliferative capacity of one or more cells. A cell has reducedproliferative capacity when the cell becomes less able to proliferate,for example, because the cell undergoes apoptosis or otherwise dies, thecell fails to proceed through the cell cycle and/or fails to divide, thecell differentiates, etc. Nonlimiting exemplary cytotoxic agentsinclude, but are not limited to, radioisotopes, toxins, andchemotherapeutic agents. One skilled in the art can select a suitablecytotoxic according to the intended application. In some embodiments,the cytotoxic agent is at least one of an anti-metabolite, an alkylatingagent, an antibiotic, a growth factor, a cytokine, an anti-angiogenicagent, an anti-mitotic agent, an anthracycline, toxin, or an apoptoticagent

In some embodiments, a label and/or a cytotoxic agent is conjugated toan antibody using chemical methods in vitro. Nonlimiting exemplarychemical methods of conjugation are known in the art, and includeservices, methods and/or reagents commercially available from, forexample, Thermo Scientific Life Science Research Produces (formerlyPierce; Rockford, Ill.), Prozyme (Hayward, Calif.), SACRI AntibodyServices (Calgary, Canada), AbD Serotec (Raleigh, N.C.), etc. In someembodiments, when a label and/or cytotoxic agent is a polypeptide, thelabel and/or cytotoxic agent can be expressed from the same expressionvector with at least one antibody chain to produce a polypeptidecomprising the label and/or cytotoxic agent fused to an antibody chain.One skilled in the art can select a suitable method for conjugating alabel and/or cytotoxic agent to an antibody according to the intendedapplication.

In some embodiments, conjugation can be covalent. In some embodiments,conjugation can be non-covalent. In some embodiments, conjugation can bevia a specific binding interaction, for example, through the binding ofa secondary antibody.

Exemplary Leader Sequences

In order for some secreted proteins to express and secrete in largequantities, a leader sequence from a heterologous protein may bedesirable. In some embodiments, employing heterologous leader sequencescan be advantageous in that a resulting mature polypeptide can remainunaltered as the leader sequence is removed in the ER during thesecretion process. The addition of a heterologous leader sequence can beuseful to express and secrete some proteins.

Certain exemplary leader sequence sequences are described, for example,in the online Leader sequence Database maintained by the Department ofBiochemistry, National University of Singapore. See Choo et al., 2005,BMC Bioinformatics, 6: 249; and PCT Publication No. WO 2006/081430.

III. Antibody Activity

Provided herein are anti-LILRB2 antibodies that provide specificfunctional characteristics. In particular aspects of the invention,anti-LILRB2 antibodies promote immunogenicity (e.g., as exhibited byM1-like macrophages) to respond to a pathology, e.g., cancer.Additionally or alternatively, anti-LILRB2 antibodies of the inventioncan inhibit an immunoregulatory (e.g., immunosuppressive) response,e.g., as exhibited by M2-like macrophages.

Blocking of HLA-A2 can be a model for disrupting the binding betweenLILRB2 and classical MHC class I molecules. Thus, in some embodiments ofthe invention, anti-LILRB2 antibodies block the binding of HLA-G orHLA-A2 to LILRB2 (e.g., human LILRB2). Blocking can be detected and/orquantified by any suitable means known in the art or described herein.For example, blocking of HLA-G or HLA-A2 can be detected and/orquantified using a tetramer blocking assay (e.g., using humanmonocytes), as described, e.g., in Example 10.

In some embodiments, an anti-LILRB2 antibody that blocks the binding ofHLA-G to LILRB2 binds the same epitope (i.e., wholly or partially) ofLILRB2 as HLA-G. In some embodiments, an anti-LILRB2 antibody thatblocks the binding of HLA-G to LILRB2 binds at least one, at least two,at least three, at least four, at least five, at least six, at leastseven, or at least eight residues of the LILRB2 epitope bound by HLA-G.In some embodiments, an anti-LILRB2 antibody blocks at least 50% (e.g.,from 50-100%, from 55-95%, from 60-90%, from 65-85%, or from 70-80%,e.g., from 50-60%, from 60-70%, from 70-80%, from 80-90%, or from90-100%, e.g., about 50%, about 55%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%) ofHLA-G tetramer in a tetramer binding assay. In some embodiments, ananti-LILRB2 antibody blocks HLA-G tetramer at an EC₅₀ of less than 1.0nM (e.g., less than 0.9 nM, less than 0.8 nM, less than 0.7 nM, lessthan 0.6 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, lessthan 0.2 nM, less than 0.15 nM, less than 0.14 nM, less than 0.13 nM,less than 0.12 nM, less than 0.11 nM, less than 0.1 nM, less than 0.09nM, or less than 0.08 nM) in a tetramer binding assay.

In some embodiments, an anti-LILRB2 antibody that blocks the binding ofHLA-A2 to LILRB2 binds the same epitope (i.e., wholly or partially) ofLILRB2 as HLA-A2. In some embodiments, an anti-LILRB2 antibody thatblocks the binding of HLA-A2 to LILRB2 binds at least one, at least two,at least three, at least four, at least five, at least six, at leastseven, or at least eight residues of the LILRB2 epitope bound by HLA-A2.In some embodiments, an anti-LILRB2 antibody blocks at least 50% (e.g.,from 50-100%, from 55-95%, from 60-90%, from 65-85%, or from 70-80%,e.g., from 50-60%, from 60-70%, from 70-80%, from 80-90%, or from90-100%, e.g., about 50%, about 55%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%) ofHLA-A2 tetramer in a tetramer binding assay. In some embodiments, ananti-LILRB2 antibody blocks HLA-A2 tetramer at an EC₅₀ of less than 1.0nM (e.g., less than 0.9 nM, less than 0.8 nM, less than 0.7 nM, lessthan 0.6 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, lessthan 0.2 nM, less than 0.15 nM, less than 0.14 nM, less than 0.13 nM,less than 0.12 nM, less than 0.11 nM, less than 0.1 nM, less than 0.09nM, or less than 0.08 nM) in a tetramer binding assay.

In some embodiments, anti-LILRB2 antibodies provided herein are capableof converting an M2-like macrophage population to an M1-like macrophagepopulation. Conversion of an M2-like macrophage to an M1-like macrophagecan be detected or quantified using any suitable method known in the artor described herein, e.g., a human monocyte-derive macrophage assay asdescribed in Example 6 or a histoculture assay as described in Example13.

In some embodiments, the conversion of an M2-like macrophage to anM1-like macrophage is indicated by an increased expression of one ormore genes selected from the group consisting of CXCL9, CXCL11, IRF1,TAP1, IL6R, and IL15, e.g., an increase of expression of at least 1%(e.g., at least 2%, at least 3%, at least 4%, at least 5%, at least 10%,at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, atleast 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, atleast 40-fold, at least 50-fold, at least 100-fold, or more) of any oneor more genes selected from the group consisting of CXCL9, CXCL11, IRF1,TAP1, IL6R, and IL15. In some embodiments, the conversion of an M2-likemacrophage to an M1-like macrophage is indicated by a decreasedexpression of one or more genes selected from the group consisting ofCCL2, PTPN22, KLRC3, IL10, IL18R1, G6PD, CD68, and BAT3, e.g., adecrease of expression of at least 1% (e.g., at least 2%, at least 3%,at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, or 100%) of any one or more genesselected from the group consisting of CCL2, PTPN22, KLRC3, IL10, IL18R1,G6PD, CD68, and BAT3.

In some embodiments, the conversion of an M2-like macrophage to anM1-like macrophage is indicated by an increased expression of one, two,or all three cytokines selected from the group consisting of TNFα, IL-6,CCL3, EGR2, TRAF1, IL1A, IRAK2, TNFalpha, IL7R, CCL2, IL8, CCL4, CXCL1,BCL2, EGR1, IL1RN, TNFSF15, DUSP4, ICAM1, TNFAIP3, TNFRSF9, CD83,TNFAIP6, CCL20, NFKB1, TNFRSF4, CXCL2, PTGS2, NFKBIA, NFKB2, CLEC4E,NFKBIZ, CCL5, CCL7, CLEC5A, CEBPB, TLR2, SRC, RELB, PLAUR, SOCS3, GBP1,CCL18, CSF1, CD40, NT5E, CCL23, CCL8, GBP5, ITGAX, C3, TNFSF15, ICAM5,DPP4, ZEB1, SPP1, IL23A, CD123, and IL6, e.g., an increase of expressionof at least 1% (e.g., at least 2%, at least 3%, at least 4%, at least5%, at least 10%, at least 15%, at least 20%, at least 25%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 1-fold, at least 1.5-fold, at least 2-fold,at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, atleast 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, atleast 100-fold, or more) of any one or more genes selected from thegroup consisting of TNFα, IL-6, CCL3, EGR2, TRAF1, ILIA, IRAK2, TNF,IL7R, CCL2, IL8, CCL4, CXCL1, BCL2, EGR1, IL1RN, TNFSF15, DUSP4, ICAM1,TNFAIP3, TNFRSF9, CD83, TNFAIP6, CCL20, NFKB1, TNFRSF4, CXCL2, PTGS2,NFKBIA, NFKB2, CLEC4E, NFKBIZ, CCL5, CCL7, CLEC5A, CEBPB, TLR2, SRC,RELB, PLAUR, SOCS3, GBP1, CCL18, CSF1, CD40, NT5E, CCL23, CCL8, GBP5,ITGAX, C3, TNFSF15, ICAM5, DPP4, ZEB1, SPP1, IL23A, CD123, and IL6. Insome embodiments, the conversion of an M2-like macrophage to an M1-likemacrophage is indicated by a decreased expression of IL-10, CCL2,TGFBR2, CXCL13, IL21R, CD36, CR1, C1QB, and TGFBI, e.g., a decrease ofexpression of at least 1% (e.g., at least 2%, at least 3%, at least 4%,at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, or 100%) of any one or more genes selected fromthe group consisting of IL-10, CCL2, TGFBR2, CXCL13, IL21R, CD36, CR1,C1QB, and TGFBI.

In some embodiments, the anti-LILRB2 antibody provided herein binds tohuman LILRB2 with a greater affinity than to any one or more of humanLILRB1, human LILRB3, human LILRB4, human LILRB5, human LILRA1, humanLILRA2, human LILRA3, human LILRA4, human LILRA5, or human LILRA6. Insome embodiments, the anti-LILRB2 antibody of the invention binds tohuman LILRB2 with at least 2-fold greater affinity (e.g., at least3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least100-fold, or more) greater affinity relative to any one or more of humanLILRB1, human LILRB3, human LILRB4, human LILRB5, human LILRA1, humanLILRA2, human LILRA3, human LILRA4, human LILRA5, or human LILRA6. Insome embodiments, binding of the anti-LILRB2 antibody provided herein toany one or more of human LILRB1, human LILRB3, human LILRB4, humanLILRB5, human LILRA1, human LILRA2, human LILRA3, human LILRA4, humanLILRA5, or human LILRA6 is undetectable, e.g., bio-layer interferometry(e.g., less than 0.08 nm by OCTET®). In some embodiments, the K_(D) ofthe anti-LILRB2 antibody provided herein to any one or more of LILRB1,human LILRB3, human LILRB4, human LILRB5, human LILRA1, human LILRA2,human LILRA3, human LILRA4, human LILRA5, or human LILRA6 is greaterthan 10 nM (e.g., greater than 15 nM, greater than 20 nM, greater than25 nM, greater than 30 nM, greater than 35 nM, greater than 40 nM,greater than 45 nM, greater than 50 nM, greater than 60 nM, greater than70 nM, greater than 80 nM, greater than 90 nM, greater than 100 nM,greater than 500 nM, greater than 1 μM, greater than 10 μM, or greaterthan 100 μM).

IV. Antibody Expression and Production

Nucleic Acid Molecules Encoding Anti-LILRB2 Antibodies Nucleic acidmolecules comprising polynucleotides that encode one or more chains ofan anti-LILRB2 antibody are provided herein. In some embodiments, anucleic acid molecule comprises a polynucleotide that encodes a heavychain or a light chain of an anti-LILRB2 antibody. In some embodiments,a nucleic acid molecule comprises both a polynucleotide that encodes aheavy chain and a polynucleotide that encodes a light chain, of ananti-LILRB2 antibody. In some embodiments, a first nucleic acid moleculecomprises a first polynucleotide that encodes a heavy chain and a secondnucleic acid molecule comprises a second polynucleotide that encodes alight chain.

In some embodiments, the heavy chain and the light chain are expressedfrom one nucleic acid molecule, or from two separate nucleic acidmolecules, as two separate polypeptides. In some embodiments, such aswhen an antibody is an scFv, a single polynucleotide encodes a singlepolypeptide comprising both a heavy chain and a light chain linkedtogether.

In some embodiments, a polynucleotide encoding a heavy chain or lightchain of an anti-LILRB2 antibody comprises a nucleotide sequence thatencodes at least one of the CDRs provided herein. In some embodiments, apolynucleotide encoding a heavy chain or light chain of an anti-LILRB2antibody comprises a nucleotide sequence that encodes at least 3 of theCDRs provided herein. In some embodiments, a polynucleotide encoding aheavy chain or light chain of an anti-LILRB2 antibody comprises anucleotide sequence that encodes at least 6 of the CDRs provided herein.In some embodiments, a polynucleotide encoding a heavy chain or lightchain of an anti-LILRB2 antibody comprises a nucleotide sequence thatencodes a leader sequence, which, when translated, is located at the Nterminus of the heavy chain or light chain. As discussed above, theleader sequence may be the native heavy or light chain leader sequence,or may be another heterologous leader sequence.

In some embodiments, the nucleic acid is one that encodes for any of theamino acid sequences for the antibodies in the Sequence Table herein. Insome embodiments, the nucleic acid is one that is at least 80% identicalto a nucleic acid encoding any of the amino acid sequences for theantibodies in the Sequence Table herein, for example, at least 80, 85,90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical. In someembodiments, the nucleic acid is one that hybridizes to any one or moreof the nucleic acid sequences provided herein. In some of theembodiments, the hybridization is under moderate conditions. In someembodiments, the hybridization is under highly stringent conditions,such as: at least about 6×SSC and 1% SDS at 65° C., with a first washfor 10 minutes at about 42° C. with about 20% (v/v) formamide in0.1×SSC, and with a subsequent wash with 0.2×SSC and 0.1% SDS at 65° C.

Nucleic acid molecules can be constructed using recombinant DNAtechniques conventional in the art. In some embodiments, a nucleic acidmolecule is an expression vector that is suitable for expression in aselected host cell.

Vectors

Vectors comprising polynucleotides that encode anti-LILRB2 heavy chainsand/or anti-LILRB2 light chains are provided. Vectors comprisingpolynucleotides that encode anti-LILRB2 heavy chains and/or anti-LILRB2light chains are also provided. Such vectors include, but are notlimited to, DNA vectors, phage vectors, viral vectors, retroviralvectors, etc. In some embodiments, a vector comprises a firstpolynucleotide sequence encoding a heavy chain and a secondpolynucleotide sequence encoding a light chain. In some embodiments, theheavy chain and light chain are expressed from the vector as twoseparate polypeptides. In some embodiments, the heavy chain and lightchain are expressed as part of a single polypeptide, such as, forexample, when the antibody is an scFv.

In some embodiments, a first vector comprises a polynucleotide thatencodes a heavy chain and a second vector comprises a polynucleotidethat encodes a light chain. In some embodiments, the first vector andsecond vector are transfected into host cells in similar amounts (suchas similar molar amounts or similar mass amounts). In some embodiments,a mole- or mass-ratio of between 5:1 and 1:5 of the first vector and thesecond vector is transfected into host cells. In some embodiments, amass ratio of between 1:1 and 1:5 for the vector encoding the heavychain and the vector encoding the light chain is used. In someembodiments, a mass ratio of 1:2 for the vector encoding the heavy chainand the vector encoding the light chain is used.

In some embodiments, a vector is selected that is optimized forexpression of polypeptides in CHO or CHO-derived cells, or in NSO cells.Exemplary such vectors are described, for example, in Running Deer etal., 2004, Biotechnol. Prog. 20:880-889.

Host Cells

In some embodiments, anti-LILRB2 antibody heavy chains and/oranti-LILRB2 antibody light chains may be expressed in prokaryotic cells,such as bacterial cells; or in eukaryotic cells, such as fungal cells(such as yeast), plant cells, insect cells, and mammalian cells. Suchexpression may be carried out, for example, according to proceduresknown in the art. Exemplary eukaryotic cells that may be used to expresspolypeptides include, but are not limited to, COS cells, including COS 7cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S,DG44. Lec13 CHO cells, and FUT8 CHO cells; PER.C6® cells (Crucell); andNSO cells. In some embodiments, anti-LILRB2 antibody heavy chains and/oranti-LILRB2 antibody light chains may be expressed in yeast. See, forexample, U.S. Publication No. U.S. 2006/0270045 A1. In some embodiments,a particular eukaryotic host cell is selected based on its ability tomake desired post-translational modifications to the anti-LILRB2antibody heavy chains and/or anti-LILRB2 antibody light chains. Forexample, in some embodiments, CHO cells produce polypeptides that have ahigher level of sialylation than the same polypeptide produced in 293cells.

Introduction of one or more nucleic acids into a desired host cell maybe accomplished by any method, including but not limited to, calciumphosphate transfection, DEAE-dextran mediated transfection, cationiclipid-mediated transfection, electroporation, transduction, infection,etc. Nonlimiting exemplary methods are described, for example, inSambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed. ColdSpring Harbor Laboratory Press (2001). Nucleic acids may be transientlyor stably transfected in the desired host cells, according to anysuitable method.

Host cells comprising any of the polynucleotides or vectors describedherein are also provided. In some embodiments, a host cell comprising ananti-LILRB2 antibody is provided. Any host cells capable ofover-expressing heterologous DNAs can be used for the purpose ofisolating the genes encoding the antibody, polypeptide or protein ofinterest. Non-limiting examples of mammalian host cells include but notlimited to COS, HeLa, and CHO cells. See also PCT Publication No. WO87/04462. Suitable non-mammalian host cells include prokaryotes (such asE. coli or B. subtillis) and yeast (such as S. cerevisae, S. pombe; orK. lactis).

Purification of Antibodies

Anti-LILRB2 antibodies can be purified by any suitable method. Suchmethods include, but are not limited to, the use of affinity matrices orhydrophobic interaction chromatography. Suitable affinity ligandsinclude the ROR1 ECD and ligands that bind antibody constant regions.For example, a Protein A, Protein G, Protein A/G, or an antibodyaffinity column may be used to bind the constant region and to purify ananti-LILRB2 antibody. Hydrophobic interactive chromatography, forexample, a butyl or phenyl column, may also suitable for purifying somepolypeptides such as antibodies. Ion exchange chromatography (forexample anion exchange chromatography and/or cation exchangechromatography) may also suitable for purifying some polypeptides suchas antibodies. Mixed-mode chromatography (for example reversedphase/anion exchange, reversed phase/cation exchange, hydrophilicinteraction/anion exchange, hydrophilic interaction/cation exchange,etc.) may also suitable for purifying some polypeptides such asantibodies. Many methods of purifying polypeptides are known in the art.

Cell-Free Production of Antibodies

In some embodiments, an anti-LILRB2 antibody is produced in a cell-freesystem. Nonlimiting exemplary cell-free systems are described, forexample, in Sitaraman et al., 2009, Methods Mol. Biol. 498: 229-44;Spirin, 2004, Trends Biotechnol. 22: 538-45; Endo et al., 2003,Biotechnol. Adv. 21: 695-713.

Compositions

In some embodiments, antibodies prepared by the methods described aboveare provided. In some embodiments, the antibody is prepared in a hostcell. In some embodiments, the antibody is prepared in a cell-freesystem. In some embodiments, the antibody is purified. In someembodiments, the antibody prepared in a host cell or a cell-free systemis a chimeric antibody. In some embodiments, the antibody prepared in ahost cell or a cell-free system is a humanized antibody. In someembodiments, the antibody prepared in a host cell or a cell-free systemis a human antibody. In some embodiments, a cell culture mediacomprising an anti-LILRB2 antibody is provided. In some embodiments, ahost cell culture fluid comprising an anti-LILRB2 antibody is provided.

In some embodiments, compositions comprising antibodies prepared by themethods described above are provided. In some embodiments, thecomposition comprises an antibody prepared in a host cell. In someembodiments, the composition comprises an antibody prepared in acell-free system. In some embodiments, the composition comprises apurified antibody. In some embodiments, the composition comprises achimeric antibody prepared in a host cell or a cell-free system. In someembodiments, the composition comprises a humanized antibody prepared ina host cell or a cell-free system. In some embodiments, the compositioncomprises a human antibody prepared in a host cell or a cell-freesystem.

In some embodiments, a composition comprising anti-LILRB2 antibody at aconcentration of more than about any one of 10 mg/mL, 20 mg/mL, 30mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, 100mg/mL, 125 mg/mL, 150 mg/mL, 175 mg/mL, 200 mg/mL, 225 mg/mL, or 250mg/mL is provided. In some embodiments, the composition comprises achimeric antibody prepared in a host cell or a cell-free system. In someembodiments, the composition comprises a humanized antibody prepared ina host cell or a cell-free system. In some embodiments, the compositioncomprises a human antibody prepared in a host cell or a cell-freesystem.

V. Therapeutic Compositions and Methods Methods of Treating DiseasesUsing Anti-LILRB2 Antibodies

Antibodies and compositions comprising antibodies are provided for usein methods of treatment for humans or animals. Methods of treatingdisease comprising administering anti-LILRB2 antibodies are alsoprovided. Nonlimiting exemplary diseases that can be treated withanti-LILRB2 antibodies include, but are not limited to cancer.

In more detail, examples of diseases, such as cancer, that can betreated according to the methods of the invention include solid andhematological/lymphatic cancers and also malignant, pre-malignant, andbenign growth, such as dysplasia. Examples of cancer include but are notlimited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular non-limiting examples of such cancers include kidney cancer(e.g., renal cell carcinoma, e.g., papillary renal cell carcinoma),squamous cell cancer, mesothelioma, teratoma, small-cell lung cancer,pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma,lung cancer (e.g., non-small cell lung cancer, adenocarcinoma of thelung, squamous carcinoma of the lung), cancer of the peritoneum,hepatocellular cancer, gastrointestinal cancer (e.g., stomach cancer),pancreatic cancer, cervical cancer, ovarian cancer, liver cancer,bladder cancer, hepatoma, breast cancer, colon cancer, colorectalcancer, rectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, liver cancer, prostate cancer, vulval cancer, thyroid cancer,thymoma, hepatic carcinoma, brain cancer, glioma, glioblastoma,endometrial cancer, testis cancer, cholangiocarcinoma, cholangiosarcoma,gallbladder carcinoma, gastric cancer, melanoma (e.g., uveal melanoma),pheochromocytoma, paraganglioma, adenoid cystic carcinoma, and varioustypes of head and neck cancer (e.g., squamous head and neck cancer).

The anti-LILRB2 antibody can be administered as needed to subjects.Determination of the frequency of administration can be made by personsskilled in the art, such as an attending physician based onconsiderations of the condition being treated, age of the subject beingtreated, severity of the condition being treated, general state ofhealth of the subject being treated and the like. In some embodiments,an effective dose of an anti-LILRB2 antibody is administered to asubject one or more times. In some embodiments, an effective dose of ananti-LILRB2 antibody is administered to the subject once a month, lessthan once a month, such as, for example, every two months or every threemonths. In some embodiments, an effective dose of an anti-LILRB2antibody is administered less than once a month, such as, for example,every two weeks or every week. An effective dose of an anti-LILRB2antibody is administered to the subject at least once. In someembodiments, the effective dose of an anti-LILRB2 antibody may beadministered multiple times, including for periods of at least a month,at least six months, or at least a year.

In some embodiments, pharmaceutical compositions are administered in anamount effective for treatment of (including prophylaxis of) cancer. Thetherapeutically effective amount is typically dependent on the weight ofthe subject being treated, his or her physical or health condition, theextensiveness of the condition to be treated, or the age of the subjectbeing treated. In general, anti-LILRB2 antibodies may be administered inan amount in the range of about 10 μg/kg body weight to about 100 mg/kgbody weight per dose. In some embodiments, anti-LILRB2 antibodies may beadministered in an amount in the range of about 50 μg/kg body weight toabout 5 mg/kg body weight per dose. In some embodiments, anti-LILRB2antibodies may be administered in an amount in the range of about 100μg/kg body weight to about 10 mg/kg body weight per dose. In someembodiments, anti-LILRB2 antibodies may be administered in an amount inthe range of about 100 μg/kg body weight to about 20 mg/kg body weightper dose. In some embodiments, anti-LILRB2 antibodies may beadministered in an amount in the range of about 0.5 mg/kg body weight toabout 20 mg/kg body weight per dose.

In some embodiments, pharmaceutical compositions are administered in anamount effective to cause conversion of M2-like macrophages to M1-likemacrophages.

The therapeutically effective amount is typically dependent on theweight of the subject being treated, his or her physical or healthcondition, the extensiveness of the condition to be treated, or the ageof the subject being treated. In general, anti-LILRB2 antibodies may beadministered in an amount in the range of about 10 μg/kg body weight toabout 100 mg/kg body weight per dose. In some embodiments, anti-LILRB2antibodies may be administered in an amount in the range of about 50μg/kg body weight to about 5 mg/kg body weight per dose. In someembodiments, anti-LILRB2 antibodies may be administered in an amount inthe range of about 100 μg/kg body weight to about 10 mg/kg body weightper dose. In some embodiments, anti-LILRB2 antibodies may beadministered in an amount in the range of about 100 μg/kg body weight toabout 20 mg/kg body weight per dose. In some embodiments, anti-LILRB2antibodies may be administered in an amount in the range of about 0.5mg/kg body weight to about 20 mg/kg body weight per dose.

Pharmaceutical Compositions

In some embodiments, compositions comprising anti-LILRB2 antibodies areprovided in formulations with a wide variety of pharmaceuticallyacceptable carriers (see, for example, Gennaro, Remington: The Scienceand Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus,20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and DrugDelivery Systems, 7th ed., Lippencott Williams and Wilkins (2004); Kibbeet al., Handbook of Pharmaceutical Excipients, 3rd ed., PharmaceuticalPress (2000)). Various pharmaceutically acceptable carriers, whichinclude vehicles, adjuvants, and diluents, are available. Moreover,various pharmaceutically acceptable auxiliary substances, such as pHadjusting and buffering agents, tonicity adjusting agents, stabilizers,wetting agents and the like, are also available. Non-limiting exemplarycarriers include saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof.

In some embodiments, a pharmaceutical composition comprising ananti-LILRB2 antibody is provided. In some embodiments, thepharmaceutical composition comprises a chimeric antibody. In someembodiments, the pharmaceutical composition comprises a humanizedantibody. In some embodiments, the pharmaceutical composition comprisesan antibody prepared in a host cell or cell-free system as describedherein. In some embodiments, the pharmaceutical composition comprisespharmaceutically acceptable carrier.

In some embodiments, pharmaceutical compositions are administered in anamount effective for treatment of (including prophylaxis of) cancer. Thetherapeutically effective amount is typically dependent on the weight ofthe subject being treated, his or her physical or health condition, theextensiveness of the condition to be treated, or the age of the subjectbeing treated. In general, anti-LILRB2 antibodies may be administered inan amount in the range of about 0.05 mg/kg body weight to about 100mg/kg body weight per dose. In some embodiments, anti-LILRB2 antibodiesmay be administered in an amount in the range of about 10 μg/kg bodyweight to about 100 mg/kg body weight per dose. In some embodiments,anti-LILRB2 antibodies may be administered in an amount in the range ofabout 50 μg/kg body weight to about 5 mg/kg body weight per dose. Insome embodiments, anti-LILRB2 antibodies may be administered in anamount in the range of about 100 μg/kg body weight to about 10 mg/kgbody weight per dose. In some embodiments, anti-LILRB2 antibodies may beadministered in an amount in the range of about 100 μg/kg body weight toabout 20 mg/kg body weight per dose. In some embodiments, anti-LILRB2antibodies may be administered in an amount in the range of about 0.5mg/kg body weight to about 20 mg/kg body weight per dose. In someembodiments, anti-LILRB2 antibodies may be administered in an amount inthe range of about 0.5 mg/kg body weight to about 10 mg/kg body weightper dose. In some embodiments, anti-LILRB2 antibodies may beadministered in an amount in the range of about 0.05 mg/kg body weightto about 20 mg/kg body weight per dose. In some embodiments, anti-LILRB2antibodies may be administered in an amount in the range of about 0.05mg/kg body weight to about 10 mg/kg body weight per dose. In someembodiments, anti-LILRB2 antibodies may be administered in an amount inthe range of about 5 mg/kg body weight or lower, for example less than4, less than 3, less than 2, or less than 1 mg/kg of the antibody.

In some embodiments, anti-LILRB2 antibodies can be present in an amountin the range of about 50 μg/kg body weight to about 5 mg/kg body weightper dose. For example, in some embodiments, a dose for a 20 kg personcan be within a range of about 1 mg to about 100 mg. In someembodiments, the dose can be within a range of 2 mg to 200 mg of theanti-LILRB2 antibody. In some embodiments, the dose can be within arange of 10 mg to 400 mg of the anti-LILRB2 antibody.

Routes of Administration

In some embodiments, anti-LILRB2 antibodies can be administered in vivoby various routes, including, but not limited to, intravenous,intra-arterial, parenteral, intratumoral, intraperitoneal orsubcutaneous. The appropriate formulation and route of administrationmay be selected according to the intended application.

Combination Therapy

Anti-LILRB2 antibodies can be administered alone or with other modes oftreatment, e.g., with an additional therapeutic agent. They can beprovided before, substantially contemporaneous with, or after othermodes of treatment, for example, surgery, chemotherapy, radiationtherapy, or the administration of a biologic, such as anothertherapeutic antibody. In some embodiments, an anti-LILRB2 antibody isadministered in conjunction with another anti-cancer agent.

In some embodiments, an anti-LILRB2 antibody provided herein isadministered with a PD-1 therapy. Exemplary PD-1 therapies include, butare not limited to, nivolumab (BMS-936558, MDX-1106, ONO-4538);pidilizumab, lambrolizumab/pembrolizumab (KEYTRUDA, MK-3475);durvalumab; RG-7446; avelumab (MSB-0010718C); AMP-224; BMS-936559 (ananti-PD-L1 antibody); AMP-514; MDX-1105; ANB-011; anti-LAG-3/PD-1;anti-PD-1 Ab (CoStim); anti-PD-1 Ab (Kadmon Pharm.); anti-PD-1 Ab(Immunovo); anti-TIM-3/PD-1 Ab (AnaptysBio); anti-PD-L1 Ab(CoStim/Novartis); MEDI-4736 (an anti-PD-L1 antibody,Medimmune/AstraZeneca); RG7446/MPDL3280A (an anti-PD-L1 antibody,Genentech/Roche); K_(D)-033, PD-1 antagonist (Agenus); STI-A1010;STI-A1110; TSR-042; and other antibodies and other agents that aredirected against programmed death-1 (PD-1) or programmed death ligand 1(PD-L1) (e.g., JTX-4014, US 2018/0118829).

In some embodiments, a subject is selected for treatment with ananti-LILRB2 antibody provided herein and a PD-1 therapy if the subject'stumor is PD-L1^(HIGH). Determining the level of PD-L1 may be determined,for example, using IHC. In some embodiments, a subject is first treatedwith a PD-1 therapy, and is later treated with an anti-LILRB2 antibodyprovided herein, with or without continuing the PD-1 therapy. Thus,methods provided herein include treatment of a subject with ananti-LILRB2 antibody, wherein the subject has previously been treatedwith a PD-1 therapy.

In some embodiments, an anti-LILRB2 antibody provided herein isadministered to patients who show the presence of macrophages in one ormore tumors. The presence of macrophages can be determined by, e.g.,mRNA signature or IHC.

In some embodiments, an anti-LILRB2 antibody provided herein isadministered with one or more therapies selected from: an anti-CD47antibody (e.g., CC90002 (Celgene) or Hu5F9-G4 (Forty Seven, Inc.)); ananti-SIRP alpha antibody (e.g., OSE-172 (OSE Immunotherapuetics));pegylated IL-2 (e.g., NKTR-214 (Nektar Therapeutics)); an anti-VEGFantibody (e.g., bevacizumab (AVASTIN®)); TTI-621 or TTI-624 (TrilliumTherapeutics SIRPa-Fc); ALX148 (Alexo, SIRPa-Fc), and an IDO inhibitor(e.g., epacadostat (Incyte)).

In some embodiments, a subject is selected for treatment with ananti-LILRB2 antibody provided herein and an ICOS therapy (e.g.,JTX-2011, e.g., as described in U.S. Patent Publication No.2016/0304610, incorporated herein by reference in its entirety). In someembodiments, a subject is first treated with an ICOS therapy, and islater treated with an anti-LILRB2 antibody provided herein, with orwithout continuing the ICOS therapy. Thus, methods provided hereininclude treatment of a subject with an anti-LILRB2 antibody, wherein thesubject has previously been treated with an ICOS therapy.

In some embodiments, the anti-LILRB2 antibody provided herein isadministered with an agonist anti-OX40 antibody (such as Medi6469,MedImmune; MOXR0916/RG7888, Roche). In some embodiments, the anti-LILRB2antibody provided herein is administered with an anti-CTLA4 antibody(such as ipilimumab, YERVOY®, BMS-734016; MDX-101).

In some embodiments, an additional therapeutic agent is achemotherapeutic agent. Exemplary chemotherapeutic agents that may becombined with the anti-LILRB2 antibodies provided herein include, butare not limited to, capectiabine, cyclophosphamide, dacarbazine,temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin,cisplatin, carboplatin, epirubicin, eribulin, 5-FU, gemcitabine,irinotecan, ixabepilone, methotrexate, mitoxantrone, oxaliplatin,paclitaxel, nab-paclitaxel, ABRAXANE® (protein-bound paclitaxel),pemetrexed, vinorelbine, and vincristine. In some embodiments, ananti-LILRB2 antibody provided herein is administered with at least onekinase inhibitor. Nonlimiting exemplary kinase inhibitors includeerlotinib, afatinib, gefitinib, crizotinib, dabrafenib, trametinib,vemurafenib, and cobimetanib.

In some embodiments, the additional therapeutic agent is an IDOinhibitor. Nonlimiting exemplary IDO inhibitors are described, e.g., inUS 2016/0060237; and US 2015/0352206. Nonlimiting exemplary IDOinhibitors include Indoximod (New Link Genetics), INCB024360 (IncyteCorp), 1-methyl-D-tryptophan (New Link Genetics), and GDC-0919(Genentech).

In some embodiments, an anti-LILRB2 antibody provided herein isadministered in combination with an immune-modifying drug (IMiD).Nonlimiting exemplary IMiDs include thalidomide, lenalidomide, andpomalidomide.

In some embodiments, the anti-LILRB2 antibody is administered with asecond therapeutic method for treatment. Thus, the administration of anantibody provided herein can be in combination with another system oftreatment.

In some embodiments, an additional therapeutic agent is a cancervaccine. Cancer vaccines have been investigated as a potential approachfor antigen transfer and activation of dendritic cells. In particular,vaccination in combination with immunologic checkpoints or agonists forco-stimulatory pathways have shown evidence of overcoming tolerance andgenerating increased anti-tumor response. A range of cancer vaccineshave been tested that employ different approaches to promoting an immuneresponse against the tumor (see, e.g., Emens, 2008, Expert Opin. Emerg.Drugs, 13(2): 295-308). Approaches have been designed to enhance theresponse of B cells, T cells, or professional antigen-presenting cellsagainst tumors. Exemplary types of cancer vaccines include, but are notlimited to, peptide-based vaccines that employ targeting distinct tumorantigens, which may be delivered as peptides/proteins or asgenetically-engineered DNA vectors, viruses, bacteria, or the like; andcell biology approaches, for example, for cancer vaccine developmentagainst less well-defined targets, including, but not limited to,vaccines developed from patient-derived dendritic cells, autologoustumor cells or tumor cell lysates, allogeneic tumor cells, and the like.

Nonlimiting exemplary cancer vaccines include Sipuleucel-T, which isderived from autologous peripheral-blood mononuclear cells (PBMCs) thatinclude antigen-presenting cells (see, e.g., Kantoff P W et al., 2010, NEngl J Med 363:411-22). In Sipuleucel-T generation, the patient's PBMCsare activated ex vivo with PA2024, a recombinant fusion protein ofprostatic acid phosphatase (a prostate antigen) andgranulocyte-macrophage colony-stimulating factor (an immune-cellactivator). Another approach to a candidate cancer vaccine is togenerate an immune response against specific peptides mutated in tumortissue, such as melanoma (see, e.g., Carreno et al., 2015, Science348:6236). Such mutated peptides may, in some embodiments, be referredto as neoantigens. As a nonlimiting example of the use of neoantigens intumor vaccines, neoantigens in the tumor predicted to bind the majorhistocompatibility complex protein HLA-A*02:01 are identified forindividual patients with a cancer, such as melanoma. Dendritic cellsfrom the patient are matured ex vivo, then incubated with neoantigens.The activated dendritic cells are then administered to the patient. Insome embodiments, following administration of the cancer vaccine, robustT-cell immunity against the neoantigen is detectable.

In some such embodiments, the cancer vaccine is developed using aneoantigen. In some embodiments, the cancer vaccine is a DNA vaccine,such as a mammaglobin-A DNA vaccine (see, e.g., Gillanders et al., 2014,Clin. Canc. Res., 20: 5964-75). In some embodiments, the cancer vaccineis an engineered virus comprising a cancer antigen, such as PROSTVAC(rilimogene galvacirepvec/rilimogene glafolivec). In some embodiments,the cancer vaccine comprises engineered tumor cells, such as GVAX, whichis a granulocyte-macrophage colony-stimulating factor (GM-CSF)gene-transfected tumor cell vaccine (see, e.g., Nemunaitis, 2005, ExpertRev Vaccines, 4: 259-74).

In some embodiments, an anti-LILRB2 antibody described herein isadministered before, concurrently, or after a cancer vaccine. In someembodiments, cancer vaccines developed using neoantigens are used incombination with the anti-LILRB2 antibodies described herein. In somesuch embodiments, the combination is used to treat a cancer with a highmutational burden, such as melanoma, lung, bladder, or colorectalcancer.

In some embodiments, an anti-LILRB2 antibody provided herein isadministered in combination with a chimeric antigen receptor T celltherapy (CAR-T therapy). The CAR-T cell may be genetically modified toexpress a receptor that recognizes an antigen expressed by tumor cell.The antigen may be an antigen specifically expressed by the tumor or anantigen expressed by both cancerous cells and healthy tissue. In someembodiments, the CAR-T cell is an anti-BCMA CAR-T cell. In someembodiments, CAR-T therapy is adoptive CAR-T therapy, in which apatients T cells are removed and modified to express the chimericantigen receptor, and then returned to the patient. See, e.g., Dai etal., 2016, J Natl Cancer Inst, 108 (7): djv439, doi:10.1093/jnci/djv439; Gill et al., 2015, Blood Rev,50268-960X(15)00080-6, doi: 10.1016/j.blre.2015.10.003; Gill et al.,2015, Immunol. Rev, 263(1):68-89. doi: 10.1111/imr.12243.

Kits/Articles of Manufacture

Provided herein are also kits, medicines, compositions, and unit dosageforms for use in any of the methods described herein.

Kits can include one or more containers comprising an anti-LILRB2antibody (or unit dosage forms and/or articles of manufacture). In someembodiments, a unit dosage is provided wherein the unit dosage containsa predetermined amount of a composition comprising an anti-LILRB2antibody, with or without one or more additional agents. In someembodiments, such a unit dosage is supplied in single-use prefilledsyringe for injection. In some embodiments, the composition contained inthe unit dosage can comprise saline, sucrose, or the like; a buffer,such as phosphate, or the like; and/or be formulated within a stable andeffective pH range. In some embodiments, the composition can be providedas a lyophilized powder that may be reconstituted upon addition of anappropriate liquid, for example, sterile water. In some embodiments, thecomposition comprises one or more substances that inhibit proteinaggregation, including, but not limited to, sucrose and arginine. Insome embodiments, a composition comprises heparin and/or a proteoglycan.

In some embodiments, the amount of the anti-LILRB2 antibody used in theunit dose can be any of the amounts provided herein for the variousmethods and/or compositions described.

In some embodiments, kits further comprise instructions for use in thetreatment of cancer in accordance with any of the methods describedherein. The kit may further comprise a description of selection anindividual suitable or treatment. Instructions supplied in the kits aretypically written instructions on a label or package insert (forexample, a paper sheet included in the kit), but machine-readableinstructions (for example, instructions carried on a magnetic or opticalstorage disk) are also acceptable. In some embodiments, the kit furthercomprises another therapeutic agent.

The kits are in suitable packaging. Suitable packaging includes, but isnot limited to, vials, bottles, jars, flexible packaging (for example,sealed Mylar or plastic bags), and the like. Kits may optionally provideadditional components such as buffers and interpretative information.The present application thus also provides articles of manufacture,which include vials (such as sealed vials), bottles, jars, flexiblepackaging, and the like.

EXAMPLES

The examples discussed below are intended to be purely exemplary of theinvention and should not be considered to limit the invention in anyway. The examples are not intended to represent that the experimentsbelow are all or the only experiments performed. Efforts have been madeto ensure accuracy with respect to numbers used (for example, amounts,temperature, etc.) but some experimental errors and deviations should beaccounted for. Unless indicated otherwise, temperature is in degreesCentigrade, and pressure is at or near atmospheric.

Example 1. HLA-G Cell Culture Experiments

The following example investigates the role of HLA-G in suppressingmyeloid cell function. FIG. 1 shows a model of a LILRB2-expressingmyeloid cell and an HLA-G-expressing tumor cell. A blocking anti-LILRB2antibody is depicted between the LILRB2 expressed on the myeloid celland the HLA-G expressed on the tumor cell. Multimeric HLA-G expressed astetramers were used in primary human myeloid cell assays to investigatethe role of HLA-G in suppressing dendritic cell function.

Non-adherent immature dendritic cells (iDCs) were collected and washedtwice in 1×DPBS (Gibco), and then were plated at 1×10⁵ cells per well ina 96-well round bottom tissue culture treated plate in RPM11640supplemented with GLUTAMAX™ (Gibco) and 10% HI-FBS (Sigma). iDCs wereeither plated in media alone, or matured in media containing PGE2,IL-1β, and TNFα in the presence or absence of HLA-G tetramer (FredHutchinson Cancer Research Center) and incubated at 37° C.+5% CO₂. After24 hours, supernatants were collected for cytokine analysis viacytometric bead array (CBA) according to manufacturer's protocol (BD)and analyzed using an Accuri C6 analyzer (BD). Cells were stained withantibodies specific for important antigen presentation molecules andcell expression of these markers was assessed using a FACSCELESTA™ flowcytometer analyzer (BD).

DCs matured in the presence of HLA-G tetramer exhibited a decrease inexpression of maturation markers CD80 and CD86 (FIGS. 2A-2F). Thisinhibitory effect was abolished when HLA-G tetramers were incubated withLILRB2^(low) donor dendritic cells (FIGS. 2G-2L). These resultsdemonstrate that soluble HLA-G blocks the maturation and ability fordendritic cells to develop into potent antigen presenting cells, andthat HLA-G-mediated suppression is dependent on LILRB2 expressed ondendritic cells.

Example 2. Generation of Antibodies

Mice and rats were immunized with human LILRB2 protein or cellsoverexpressing human LILRB2 with a DNA plasmid encoding for humanLILRB2. All antibodies except J-16 and J-18 to J-20 are of murineorigin; J-16 and J-18 to J-20 are derived from rat immunizations.Hybridoma clone supernatants were screened for specificity to humanLILRB2 over other LILR family members using cell lines overexpressingfull length LILR proteins. Hybridoma clones of interest were scaled upand supernatant was purified for more extensive antibody screeningbefore clones of interest were sequenced and produced recombinantly ashuman IgG4 chimeras. (See FIGS. 3A and 3B.)

Example 3. Chimeric Antibody Screening: Initial Anti-LILRB2 ScreenSet-Up

Due to the high degree of sequence similarity among the eleven reportedhuman LILR family members, antibodies were screened for specificityagainst all family members at hybridoma clone, hybridoma scale-up,chimeric, and humanized antibody stages. Specificity was checked both oncells as well as with recombinant protein using the FORTEBIO® OCTET® atthe chimeric antibody stage.

On cells, specificity was defined by antibody binding below a two-foldof isotype control cutoff. Positive control antibodies were utilized toestablish expression of family members on cell surface, and antibodieswere also evaluated in comparison to positive control.

For soluble recombinant protein assays, recombinant LILR family proteinswere expressed as 6×His and/or human Fc1 fusion proteins. Antibodieswere loaded on anti-human capture (AHC) sensors at 10 μg/mL, and sensorswere baselined in kinetics buffer. Control antibodies were similarlyloaded onto sensor. If human Fc fusion proteins were used, sensors wereblocked with human Fc protein and baselined in kinetics buffer. Sensorswere then tested for association with family member proteins at 300 nM.Binding is considered a response above 0.08 nm cutoff on the OCTET®instrument

Chimeric (hIgG4) anti-LILRB2 antibodies were selected based onspecificity to cell-expressed hLILRB2 over the ten other human LILRfamily members, ability to block the ligand interactions tocell-expressed LILRB2, and ability to convert M2-like macrophages toM1-like macrophages having an inflammatory activation status in aprimary human macrophage assay. Select LILRB2-specific, ligand-blockingantibodies were additionally screened for binding to non-human primate(NHP) monocytes. An isotype control antibody was included in all screensto determine background signals. Described below are the specificcriteria and strategy performed to identify ligand-blocking,hLILRB2-specific, chimeric antibodies. Results are summarized in FIGS.3A and 3B and described in detail below.

Antibody Binning Summary

A subset of antibodies against LILRB2 were binned using the OCTET® Red96in sandwich format. Briefly, antibody #1 (indicated in column 1) wasloaded onto an AHC sensor at 10 μg/mL. Tips were baselined in kineticsbuffer, blocked with hFc1, baselined in kinetics buffer, then loadedwith human LILRB2. Tips were again baselined in kinetics buffer andloaded with 10 μg/mL of antibody #2 (indicated in row 1). The responsevalues shown in Table 2, below, is the binding of antibody #2 in thesandwich format described.

TABLE 2 Competitive binding assay results J-17 J-19 J-11 J-03 J-16 J-07J-04 J-17 −0.11 −0.10 −0.27 0.43 0.52 0.40 0.46 J-19 −0.21 −0.21 0.280.54 0.63 0.45 0.54 J-11 −0.39 0.71 −0.24 0.51 0.65 0.47 0.54 J-03 0.050.15 0.01 −0.08 −0.08 −0.07 −0.07 J-16 0.09 0.47 0.30 −0.28 −0.36 −0.26−0.31 J-07 0.04 0.27 0.10 −0.29 −0.30 −0.28 −0.30 J-04 0.42 0.67 0.51−0.10 −0.10 −0.10 −0.10

Antibodies identified as specific binders to LILRB2 and potent blockersof HLA-G binding to LILRB2 (J-19, J-11, and J-17) fall in close but notentirely overlapping epitope bins. J-11 and J-19 did not block bindingof each other to LILRB2, but both were blocked by J-17. Antibodies thatare specific to LILRB2 but do not block HLA-G, J-04, J-03, and J-07,bind in a separate bin from the three antibodies that are specific andblock HLA-G, but the same bin as an antibody that blocks HLA-G bindingto LILRB2 but is cross-reactive to LILRA1, J-16. Results are shown inTable 3, below.

TABLE 3 Antibody binning results mAb1 J-17 J-19 J-11 J-03 J-16 J-07 J-04Blocked mAb J-19 J-17 J-17 J-16 J-03 J-16 J-16 Blocked mAb J-11 J-07J-07 J-03 J-03 Blocked mAb J-04 J-04 J-04 J-07 bin A/B A B C C C C

Example 4. Chimeric Antibody Screening: Screening AgainstCross-Reactivity with LILR Family Members

The purpose of this screen was to identify antibodies with specificbinding to hLILRB2 expressed on cells, with a counter-screen againstcell-expressed hLILRB1, hLILRB3, hLILRB4, hLILRB5, hLILRA1, hLILRA2,hLILRA3, hLILRA4, hLILRA5, and hLILRA6. 25 chimeric (hIgG4) antibodieswere screened for cellular hLILRB2-specificity. Positive hits forhLILRB2 binding were identified as antibodies that bound hLILRB2-CHO-sgreater than two-fold over isotype control mAb binding. Antibodies thatalso bound non-LILRB2 expressing cells greater than two-fold overisotype control mAb binding were designated as non-LILRB2-specific, orcross-reactive.

As shown in FIG. 4, 76% of chimeric antibodies tested were confirmed tobind cell-expressed human LILRB2. 78% of these hLILRB2-bindingantibodies exhibited specific binding to hLILRB2 over the other tenhLILR family members over-expressed on CHO-s. Antibodies detected with abinding of 2-fold greater than isotype control for another LILRover-expressing cell line in addition to hLILRB2 CHO-s were found to becross-reactive to hLILRB3, hLILRA1, hLILRA2, and/or hLILRA3. No hLILRB1,hLILRB4, hLILRA4, hLILRA5, or hLILRA6 cross-reactive antibodies wereidentified in this screen.

Example 5. Chimeric Antibody Screening: Screening for HLA-G BlockingAnti-LILRB2 Chimeric mAbs and HLA-A2 Blocking Anti-LILRB2 Chimeric mAbs

Antibodies were additionally screened for ability to blockligand-receptor interactions in a cell-based assay. Upon HLA-G bindingLILRB2, myeloid cells are rendered immunosuppressive. Thus, identifyinganti-LILRB2 antibodies that are capable of blocking HLA-G:LILRB2interactions are predicted to be beneficial in promoting anti-tumorresponses. In addition to HLA-G, another LILRB2 ligand capable ofsuppressing myeloid cells are classical major histocompatibility complex(MHC) class I molecules, such as HLA-A2.

In the secondary screen, 1×10⁵ CHO-s cells over-expressing human LILRB2were plated in 96-well round-bottom tissue culture-treated plate andwashed twice with 1×DPBS (Gibco) and incubated with 10 μg/mL primaryantibody (anti-LILRB2 mAbs or control) prepared in 50 μL FACS buffer(1×DPBS containing 2% HI-FBS (Sigma)+0.05% Sodium Azide). Afterincubation with mAbs for 30 minutes at 4° C., cells were washed twice inFACS buffer and then resuspended in 50 μL of FACS buffer containing 5μg/mL APC-conjugated HLA-A2 or HLA-G tetramer (Fred Hutch). Cells wereincubated protected from light for 30-60 minutes at 4° C. Afterincubation with tetramer, cells were washed in FACS buffer andre-suspended in fix buffer (1.5% paraformaldehyde diluted in 1×DPBS).Samples were analyzed using the Celesta flow cytometer analyzer (BDBiosciences). Data represent percent tetramer blocked by mAbs relativeto tetramer alone, calculated according to the following equation:

${\% \mspace{14mu} {tetramer}\mspace{14mu} {blocked}} = {100 - \left( {\frac{\left( {MFI}_{{Tetramer} + {mAb}} \right)}{{MFI}_{Tetramer}} \times 100} \right)}$

The results of a study distinguishing HLA-G blocking antibodies fromHLA-G non-blocking antibodies is shown in FIG. 5. Antibodies capable ofblocking HLA-G:LILRB2 interactions on cells is identified as the percentof tetramer bound to LILRB2+ cells in the presence of antibody comparedto tetramer bound to cells in the absence of antibody. Seven out of the25 anti-LILRB2 antibodies blocked HLA-G tetramer binding to hLILRB2+CHO-s by at least 50%. None of the isotype control antibodies blockedHLA-G from binding LILRB2+ cells.

The results of a study distinguishing HLA-A2 blocking antibodies fromHLA-A2 non-blocking antibodies is shown in FIG. 6. Antibodies capable ofblocking HLA-A2:LILRB2 interactions on cells is identified as thepercent of tetramer bound to LILRB2+ cells in the presence of antibodycompared to tetramer bound to cells in the absence of antibody. Six outof the 25 anti-LILRB2 antibodies blocked HLA-A2 tetramer binding tohLILRB2+ CHO-s by at least 50%. None of the isotype control antibodiesblocked HLA-A2 from binding LILRB2+ cells.

Example 6. Chimeric Antibody Screening: Screening Anti-LILRB2 ChimericmAbs for Biological Activity in Cell Culture Human MacrophageMonoculture-Cytokine Release Assay

Primary human monocytes from healthy donor peripheral blood weredifferentiated into macrophages in the presence of M-CSF. After sevendays of differentiation, 1×10⁵ human monocyte differentiated macrophages(HMDMs) were plated per well in a 96-well round-bottom tissue culturetreated plated in a final volume of 200 μL containing 100 ng/mL LPS inthe absence or presence of 1 μg/mL soluble mAbs in cell culture media(RPMI (Gibco)+10% FBS (Sigma)). After incubation for 24 hours at 37° C.with 5% CO₂, supernatant was collected and cytokine bead array (CBA) wasperformed according to manufacturer's protocol (BD Biosciences) tomeasure cytokines produced in response to mAbs. Samples were analyzedusing the Accuri C6 cytometer analyzer (BD Biosciences). Data representmean of two-to-four donors.

M1/inflammatory and M2/anti-inflammatory cytokine production by primaryHMDMs were detected upon treatment with soluble anti-LILRB2 mAbs, asshown in FIGS. 7A and 7B. M1/inflammatory cytokines measured includedTNFα, as well as IL-6 and IL-1β (data not shown). M2/anti-inflammatorycytokines measured included IL-10, as well as CCL-2 (data not shown).Production of cytokines is described as normalized levels relative toLPS treatment alone.

A positive correlation between M1-promoting activity (as measured byTNFα increase) and the ability for anti-LILRB2 mAbs to blockHLA-G/A:LILRB2 interactions was observed (FIGS. 8A and 8B).

Human Macrophage Monoculture-Nanostring Assay

Primary human monocytes were differentiated into macrophages in thepresence of M-CSF. After seven days of differentiation, 1×10⁵ HMDMs wereplated per well in a 96-well round-bottom tissue culture treated platein a final volume of 200 μL containing 100 ng/mL LPS in the absence orpresence of 10 μg/mL soluble anti-hLILRB2 mAbs in cell culture media(RPMI (Gibco)+10% FBS (Sigma)). Similar conditions were prepared toevaluate mAb activity in the absence of LPS. Cells were incubated at 37°C. with 5% CO₂, and separate wells were plated to assess gene changes atfour and 24 hours post-treatment. At each time point, supernatant wascollected and RNA was extracted from the cells, quantified using Quibit,and QC'd using AATI's Fragment Analyzer. If sufficient RNA was extractedfrom the sample, gene expression was performed using NanoString nCounterusing the Human Immunology V2 panel as well as a custommacrophage-specific spike-in. Gene expression was normalized to theexpression of housekeeping genes, then noise thresholding was performedusing the data from negative probes. Gene expression was transformed tothe log 2 space and data from samples that were treated with LILRB2binders were normalized to data from the palivizumab treated sample fromthe same donor. Data represent results from four donors.

Log 2(gene expression) for each donor and each treatment were normalizedto the log 2(gene expression) in response to palivizumab control. Tables4 and 5 list the differentially expressed genes, in the presence orabsence of LPS, respectively, calculated using the ttest function inMATLAB. Genes that had median log 2(fold change) across all donorseither greater than 1 (i.e. 2 fold increase) or less than −1 (i.e., 2fold decrease) with p<0.05 in response to all anti-LILRB2 antibodies inthe absence of LPS after four hours of exposure to the drugs (Table 4)and genes that were differentially expressed in response to allanti-LILRB2 antibodies in the presence of LPS after 24 hours of exposureto the drugs (Table 5). These changes were consistent across donors andare the basis for the monoculture signature scores defined in theHistoculture section.

TABLE 4 Monoculture anti-LILRB2 differential gene expression at fourhours without LPS CCL4 CCL2 TNFAIP6 CLEC4E CCL18 CASP10 CIITA IL8 IL7RBCL2 NFKB2 GBP1 CASP2 CCL3 DUSP4 CXCL2 CCL5 SOCS3 TLR7 IL1B TNFSF15CCL20 CCL7 CSF1 MAF CXCL1 IL1RN NFKB1 SRC CD40 IFI16 TRAF1 ICAM1 NFKBIACEBPB CCL23 IL16 IL1A TNFAIP3 TNFRSF4 TLR2 NTSE KLRC4 TNF CCL8 EGR1CLEC5A MBP KLRK1 IRAK2 CD83 NFKBIZ RELB TGFBR2 TLR8 EGR2 TNFRSF9 PTGS2PLAUR BLNK TNFSF10

TABLE 5 Monoculture anti-LILRB2 differential gene expression at 24 hourswith LPS IL6 IL23A C3 IL21R NTSE CXCL2 CCL4 CXCL13 IL1A ZEB1 GBP5 CD123PTGS2 SRC IL8 TNFSF15 CCL2 CCL20 TNF TGFBI IL1RN DPP4 CR1 CXCL1 ICAM5C1QB IL1B ITGAX CD36 SPP1 CCL3 TRAF5

Example 7. Chimeric Antibody Screening: Screening Anti-LILRB2 ChimericmAbs for Cross-Reactivity to Non-Human Primates Methods Primary CellBinding

LILRB2 expression in humans is restricted to innate immune cell typesincluding monocytes and neutrophils. Select HLA-G/A blocking,anti-LILRB2 mAbs capable of promoting the conversion of M2 to M1-likemacrophages were tested for potential to bind human, cyno and rhesusmonocytes in whole blood. Whole blood obtained from healthy human, cyno,and rhesus donors was obtained in sodium heparin tubes. Upon receipt,100 μL undiluted whole blood was incubated with Fc-receptor blockingreagent (TruStain, Biologend) according to manufacturer's protocol.After a 15-minute incubation, biotinylated mAbs were added to the bloodat 25 μg/mL and incubated at room temperature. After 20 minutes, dilutedstreptavidin-APC (BioLegend) and the human/NHP cross-reactiveanti-CD14-BV421 clone M5E2 (BioLegend) were added and incubated for 20minutes at room temperature. Red blood cells were lysed and samplesfixed using 1× Lyse/Fix solution (BD Biosciences) according tomanufacturer's protocol. Samples were analyzed using a Celesta flowcytometer analyzer (BD Biosciences). Monocytes were identified acrossspecies as side scatter (SSC)^(hi) CD14^(hi) cells, neutrophils wereidentified as SSC^(hi)CD14^(lo) cell, and lymphocytes were identified asSSC^(lo) CD14^(neg) cells.

On-Cell Binding to Over-Expressed Rhesus-LILRB2

Anti-hLILRB2 mAb binding to rhesus LILRB2 (LILRBb) protein was assessedby incubating LILRBb-CHOs cells with select anti-hLILRB2 mABs for 30minutes. After incubation, cells were washed and incubated withanti-hIgG-APC (Jackson Labs) according to manufacturer's protocol. Cellbinding was assessed by flow cytometry using a Celesta flow cytometeranalyzer (BD Biosciences).

Results

All anti-LILRB2 mAbs tested in this assay preferentially bound monocytesand neutrophils over lymphocytes in human whole blood (FIGS. 9A-9C). Asingle anti-LILRB2 mAb exhibited cross-species reactivity to both cynoand human, with a similar preferential binding to monocytes andneutrophils over lymphocytes. Isotype control antibodies did not bindsignificantly to any cell types in human or NHP blood. FIGS. 10A and 10Bconfirm that these results translate to cells that over-expressing NHPLILRB2 (LILRBb), such that the same anti-hLILRB2 that preferentiallycross-binds to NHP monocytes and neutrophils also binds specifically torhesus LILRB2 (LILRBb) over-expressed CHO-s in a dose-dependent mannerand does not cross-react to closely related family members in rhesusincluding LILRBa.

Example 8. Humanization, Affinity Characterization, and AssessmentStrategy for Lead Chimeric Antibodies

Lead chimeras were humanized by grafting the CDRs of lead antibodiesinto human frameworks while maintaining certain amino acids to supportloop structure and chain interface. A total of five heavy chain variableregions and five light chain variable regions were generated andexpressed in combination to create a total of 25 humanized variants inthe human IgG4 backbone. These variants were expressed as recombinantprotein and filtered based on protein titer and affinity to the humanLILRB2 target. Antibodies were further characterized for functional andbiophysical properties to narrow down the panel and select humanizedleads.

Using a Mass-2 (Sierra Sensors) high capacity amine chip preimmobilizedwith AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific antibody,humanized antibodies were captured on the anti-human surface and thenbinding to human LILRB2-His was measured by flowing six differentconcentrations from 65 to 0.27 nM of the analyte over the antibodysurface. The anti-LILRB2 surface was removed (10 mM Glycine, pH 2.0) andrecaptured between all concentrations or buffer only cycles. The datawas analyzed using the Sierra Analyzer software (version 3.1.14). Allcurves were double subtracted and fit to a 1:1 Langmuir Fit. Results areshown in Table 6, below.

TABLE 6 Binding kinetics of antibodies Antibody Reference k_(a) [1/(M ·s)] k_(d) [1/s] K_(D) [M] J-19.h 5.15 × 10⁵ 1.35 × 10⁻³ 2.62 × 10⁻⁹J-11.h 3.70 × 10⁵ 9.13 × 10⁻⁴ 2.47 × 10⁻⁹ J-17.h 1.56 × 10⁶ 4.43 × 10⁻⁴2.85 × 10⁻⁹ J-19.h1 3.91 × 10⁵ 1.07 × 10⁻³ 2.73 × 10⁻⁹ J-19.h2 5.21 ×10⁵ 2.38 × 10⁻³ 4.58 × 10⁻⁹ J-19.h3 5.18 × 10⁵ 1.01 × 10⁻³ 1.96 × 10⁻⁹J19.h4 5.13 × 10⁵ 1.20 × 10⁻³ 2.33 × 10⁻⁹

Humanized (hIgG4) anti-LILRB2 antibodies were further characterizedbased on specificity to cell-expressed hLILRB2 over the ten other humanLILR family members, ability to block the ligand interactions tocell-expressed LILRB2, and ability to convert M2-like macrophages to anM1-like inflammatory activation status in a primary human macrophageassay. Antibodies were additionally screened for binding to non-humanprimate (NHP) monocytes. Humanized variants were additionally assessedfor specific EC/IC₅₀'s for affinity to cell-expressed LILRB2, ligandblocking, and cytokine production in a primary human macrophagefunctional assay. An isotype control antibody was included in allscreens to determine background signals. EC/IC₅₀'s were calculated basedon transformed, non-normalized data using GraphPad Prism software.Results are summarized in FIGS. 11A and 11B and described in detailbelow.

Example 9. Humanized Antibody Characterization: LILR FamilyCross-Reactivity Screening

The purpose of this assessment was to verify anti-LILRB2 antibodiesmaintain specificity to hLILRB2 expressed on cells post humanization,without binding other related LILR family members including hLILRB1,hLILRB3, hLILRB4, hLILRB5, hLILRA1, hLILRA2, hLILRA3, hLILRA4, hLILRA5,and hLILRA6. Positive hits for hLILRB2 binding were identified asantibodies that bound greater than or equal to a three-fold over isotypecontrol antibody binding. None of the variants tested exceed three-foldgreater non-specific binding relative to isotype. Additionally, EC₅₀cell-based affinity measurements were determined.

Methods

To test for specificity to hLILRB2 and not towards any of the ten LILRfamily members, a multiplexed cell-based barcoding approach was used bystaining cells with Far Red and/or Violet Cell Trace dyes (ThermoScientific) according to manufacturer's protocol or left unstained. Inbrief, cells were washed twice with 1×DPBS and then incubated withdiluted dye in 1×DPBS for 20 minutes at 37° C., mixing gently every 5-10minutes. Dye labeling was quenched by adding equal volume of 100% HI-FBS(Sigma) to the cells. Note that LILRB1, LILRB2, LILRB3, LILRB4, andLILRB5 cell lines are also GFP-positive, while LILRA1, LILRA2, LILRA3,LILRA4, LILRA5, and LILRA6 cell lines are GFP-negative. The cells werethen washed twice in 1×DPBS and then all 11 cell lines were combined perwell in a 96-well round bottom tissue-culture treated plate. At least25×10³ cells of each cell-line were plated per well. Cells were thenre-suspended in primary antibody (anti-LILRB2 mAbs or control) preparedin FACS buffer (1×DPBS containing 2% HI-FBS (Sigma)+0.05% Sodium Azide).After incubation at 4° C. for 30 minutes, cells were washed twice with1×DPBS and re-suspended in anti-human IgG-PE (BioLegend) diluted 1:200in FACS buffer. Following a 30 minute incubation at 4° C., protectedfrom light, cells were washed in 1×DPBS and re-suspended in fix buffer(1.5% paraformaldehyde diluted in 1×DPBS). Samples were analyzed usingthe Celesta flow cytometer analyzer (BD Biosciences). Geometric meanfluorescence intensity (gMFI) of PE was determined for each antibodyacross each cell line. Background MFI was detected with isotype controland relative binding of antibodies tested was measured as a fold overisotype.

Results

Of the anti-hLILRB2 humanized antibodies tested for binding andspecificity to hLILRB2, all of the antibodies bound highly to hLILRB2and did not cross-bind any of the ten additional hLILR family members ina cell-based binding assay (FIG. 12). The EC₅₀ of each antibody toLILRB2-expressing CHO-s was within the sub-nanomolar range (FIG. 13).

Example 10. Humanized Antibody Characterization: Blocking of HLA-G andHLA-A2 to hLILRB2+ Cells

The potency of select humanized variants in blocking the interaction ofHLA-G and HLA-A2 with primary human macrophage-expressed hLILRB2 wereassessed. Primary human monocytes were differentiated into macrophagesin the presence of M-CSF. After seven days of differentiation, 1×10⁵HMDMs were plated in 96-well round-bottom tissue culture-treated plateand washed twice with 1×DPBS (Gibco) and then incubated with 50 μLprimary antibody (anti-LILRB2 mAbs or control) prepared in FACS buffer(1×DPBS containing 2% HI-FBS (Sigma)+0.05% Sodium Azide). Afterincubation with mAbs for 30 minutes at 4° C., cells were washed twice inFACS buffer and then resuspended in 50 μL of FACS buffer containing 5μg/mL APC-conjugated HLA-G or HLA-A2 tetramer (Fred Hutch). Cells wereincubated protected from light for 30-60 minutes at 4° C. Afterincubation with tetramer, cells were washed in FACS buffer andre-suspended in fix buffer (1.5% paraformaldehyde diluted in 1×DPBS).Samples were analyzed using the Celesta flow cytometer analyzer (BDBiosciences).

As shown in FIG. 14, all anti-hLILRB2 humanized antibodies testedblocked HLA-G interaction to cell-expressed hLILRB2 with an EC₅₀'s inthe nanomolar range. EC₅₀ values for each of the variants tested areshown in FIG. 11B. Control antibodies including isotype controls andnon-ligand blocking anti-hLILRB2 chimeric antibodies did not display anyactivity in this assay.

As shown in FIG. 15, all anti-hLILRB2 humanized antibodies testedblocked HLA-A2 interaction to cell-expressed hLILRB2 with an EC₅₀'s inthe nanomolar range. EC₅₀ values for each of the variants tested areshown in FIG. 11B.

Example 11. Humanized Antibody Characterization: Biological Activity ofHumanized Anti-LILRB2 mAbs in Cell Culture

Tumor associated macrophages (TAMs) display a functional activationstatus consistent with an M2-like, immunosuppressive macrophage. Withoutwishing to be bound by theory, antagonizing the inhibitory receptor,LILRB2, on macrophages is hypothesized to prevent the induction ofimmunosuppressive macrophages and promote hyper-inflammatory responses.To characterize the functional activity of anti-LILRB2 antibodies,EC/IC₅₀'s were determined as a potency measurement of anti-LILRB2 mAbscapable of converting M2 into M1-like macrophages in a humanmonocyte-derived macrophage (HMDM) cytokine release assay. Antibodieswith sub-nM activity (EC₅₀) in this assay were designated asM1-promoting mAbs.

Methods

After seven days of differentiation, 1×10⁵ HMDMs were plated per well ina 96-well round-bottom tissue culture treated plate in a final volume of200 μL containing 100 ng/mL LPS in the absence or presence mAbs in cellculture media (RPMI (Gibco)+10% FBS (Sigma)). After incubation for 24hours at 37° C. with 5% CO₂, supernatant was collected and CBA wasperformed according to manufacturer's protocol (BD Biosciences) tomeasure cytokines produced in response to mAbs. Samples were analyzedusing the Accuri C6 cytometer analyzer (BD Biosciences).

Results

As shown in FIGS. 16A and 16B, all anti-hLILRB2 humanized mAbs displayedM1-promoting activity, while suppressing the production M2-associatedcytokines including IL-10 and CCL-2. 67% of humanized anti-hLILRB2 mAbstested were shown to have sub-nanomolar activity in this assay. EC₅₀values for each of the variants tested are shown in FIG. 10B.

Example 12. Humanized Antibody Characterization: Selective Binding toNon-Human Primate LILRB2 (LILRBb)

To assess humanized mAbs for cross-species reactivity, hLILRB2-specific,ligand-blocking mAbs were assessed for additional selective binding toputative rhesus LILRB2 (LILRBb) over-expressed cells and not to closelyrelated NHP LILR family members.

Methods

Anti-hLILRB2 mAb binding to rhesus LILRB2 (LILRBb) protein was assessedby incubating LILRBb-CHOs cells with select anti-hLILRB2 mAbs for 30minutes. After incubation, cells were washed and incubated withanti-hIgG-APC (Jackson Labs) according to manufacturer's protocol. Cellbinding was assessed by flow cytometry using a Celesta flow cytometeranalyzer (BD Biosciences).

Results

All anti-hLILRB2 humanized mAbs bound to rhesus LILRB2 (LILRBb) indose-dependent and specific manner (FIG. 17A). These anti-hLILRB2 mAbsdid not bind the closely-related rhesus LILRBa protein expressed oncells (FIG. 17B).

Example 13. Gene Expression Analysis: Histoculture Experiments

Fresh human kidney tumor samples were obtained post-surgery. A sectionof each tumor was cut and fixed for IHC. Approximately 300-μM slices ofremaining tumor were placed in a six-well plate. Indicated treatmentswere added into the medium and plates were incubated at 37° C. Eachslice was treated with 10 μg/mL of one of six drugs for 24 hours. Thesix treatments included in the experiment are J-19, J-17, and J-11 (allLILRB2 binders and ligand blockers), J-04 (a LILRB2 binder that does notblock ligand binding), an anti-TIM3 antibody, and palivizumab, which wasused as a negative control.

Tumor slices were lysed using Qiagen's TissueLyser processor, andformalin-fixed paraffin-embedded (FFPE) samples were deparaffinized. RNAwas extracted from FFPE and fresh tumor samples, quantified usingQuibit, and, in certain cases, QC'd using AATI's Fragment Analyzer. Ifsufficient RNA was extracted from the sample, gene expression wasperformed using NanoString nCounter using the Human Immunology V2 panelas well as a custom macrophage-specific spike-in. Gene expression wasnormalized to the expression of housekeeping genes, and noisethresholding was performed using the data from negative probes. Geneexpression was transformed to the log 2 space and data from samples thatwere treated with LILRB2 binders or anti-TIM3 were normalized to datafrom the palivizumab treated sample from the same patient. FIG. 18 showschange in gene expression in response to treatment relative topalivizumab control. Differential gene expression relative topalivizumab control is quantified in Table 7, below. Each gene in thelist showed differential expression to at least two treatments with anominal p value less than 0.055. The fold change in gene expression waseither positive for all treatments or negative for all treatments.

TABLE 7 Histoculture differential gene expression Gene J-04 J-17 J-11J-19 TIM3 CD68 −0.29 −0.20 −0.24 −0.31 CXCL9 0.98 0.80 0.72 0.67 G6PD−0.21 −0.26 −0.25 −0.17 IL10 −0.41 −0.40 −0.37 −0.38 IL6R 0.33 0.45 0.360.31 ETS1 0.31 0.34 0.14 KCNJ2 0.55 0.49 0.82 MASP1 0.46 0.36 0.32 ZEB10.33 0.41 0.21 BAT3 −0.15 −0.12 CCL2 −0.50 −0.57 CLEC4A 0.28 0.29 CXCL110.93 0.78 GUSB 0.15 0.11 IFITM1 0.48 0.42 IL15 0.35 0.22 IL18R1 −0.22−0.30 IRF1 0.47 0.39 ITGA6 0.39 0.29 KLRC3 −0.44 −0.24 PIGR 0.58 0.71PTPN22 −0.62 −0.21 SDHA 0.25 0.20 SLC2A1 0.27 0.45 TAP1 0.24 0.24A hierarchical clustering heatmap showing the log 2 (fold change) inexpression of each gene (row) in each treated sample (column) is shownin FIG. 19. Each gene in the list showed differential expression to atleast two treatments with a nominal p value less than 0.055. Theexpression of Set 1 genes is generally downregulated in response totreatment (gray boxes) and the expression of Set 2 genes is generallyupregulated in response to treatment (black boxes).

A response score for each sample was calculated by adding the sum of thelog 2(fold change) of a subset of genes in Set 2 to the negative sum ofthe log 2(fold change) of a subset of genes in Set 1 and dividing by thenumber of genes included, according to the following equation:

${{PD}\mspace{14mu} {score}} = {\frac{1}{{\# {Set}\; 1\mspace{14mu} {genes}} + {\# \; {Set}\; 2\mspace{14mu} {genes}}}\left( {{\sum_{{Set}\; 2}{\log \; 2\left( {{fold}\mspace{14mu} {change}} \right)}} - {\sum_{{Set}\; 1}{\log \; 2\left( {{fold}\mspace{14mu} {change}} \right)}}} \right)}$

The response score for each donor to each ligand-blocking anti-LILRB2drug is shown in FIG. 20. Response to treatment is consistent acrosstreatments within donor, thus allowing for classification of donors bytheir response to anti-LILRB2 treatment. Donors with an average responsescore greater than 0.5 are classified as “full responders”; those withan average response score between 0.3 and 0.5 are classified as “partialresponders”; those with scores below 0.3 are classified as“non-responders.”

Monoculture signatures were derived based on the mean log 2 (foldchange) in gene expression across donors in response to anti-LILRB2ligand-blocking drugs in the absence of LPS after 4 hours and in thepresence of LPS after 24 hours (FIGS. 21A and 21B). Monoculturesignature scores were calculated for each histoculture sample treatedwith an anti-LILRB2 ligand-blocking drug by projection of the log 2(fold change) in gene expression onto the vector defined by themonoculture signature. The monoculture signature scores (four hours inthe absence of LPS and 24 hours in the presence of LPS) aresignificantly higher (p<0.01) in full responders compared to partial andnon-responders.

In sum, the monoculture results showed that LILRB2-binding drugs causemacrophages to differentially express a number of genes consistentlyacross donors. The set of genes that was modulated in this systemconstitutes a monoculture signature that incorporates both magnitude anddirectionality. To confirm that these pathways would also be modulatedin more complex systems, histoculture experiments were performed.Analysis of histoculture data shows that exposure of kidney tumor slicesto LILRB2-binding drugs results in upregulation of inflammatorychemokine expression, as well as differential expression of knownmyeloid-specific genes. The genes are co-regulated and are onlydifferentially expressed in a subset of samples. This subset of genesconstitutes the PD response signature, which is used to calculateresponse scores and classify samples based on response to drugs. Itshould be noted that donors that respond to one anti-LILRB2 druggenerally respond to all of them. Additionally, when the histoculturedata was projected onto the monoculture signature, the responders showedstatistically significant monoculture scores. Thus, the modulation ofmyeloid-specific genes was consistent with findings in in vitroexperiments, suggesting that the same biological pathways are beingaffected.

Example 14. Toxicology

Specificity of anti-LILRB2 antibodies was determined by assessingbinding of antibodies to red blood cells and platelets by flow cytometryor to serum proteins by ELISA. No off-target binding was observed inthese assays (FIG. 22).

The potential for anti-LILRB2 antibodies to elicit cytokine storm wasassessed in a human whole blood cytokine release assay using titrationsof soluble antibodies. The assay was incubated for 24 hours at 37° C.Plasma was then isolated and cytokines were measured using a 10-cytokineMSD panel. Data are mean+/−SD of three donors. As shown in FIGS.23A-23D, anti-LILRB2 antibodies did not exhibit induction of cytokinesassociated with cytokine storm (e.g., IL-4, IL-6, IL-18, or TNFα) inthis assay.

The potential for anti-LILRB2 antibodies to induce neutrophil activationwas assessed in human whole blood using titrations of solubleantibodies. The assay was incubated for two hours at 37° C. Changes inneutrophil activation markers (increase in CD11 b and decrease in CD62L)were assessed by flow cytometry. Data are mean+/−SD of 2 donors.Anti-LILRB2 antibodies did not induce neutrophil activation, asindicated by low CD11b expression (FIGS. 24A and 24B) and retention ofCD62L (FIGS. 24C and 24D).

Example 15. Pharmacokinetics

Cynomolgus monkeys (n=3 per group) received a single intravenousinfusion of the indicated concentration of anti-LILRB2 antibody. Serumconcentrations of drug were measured using an electrochemiluminescentassay, and the results, shown in FIG. 25, indicate that single-dosepharmacokinetics in cynomolgus monkeys exhibits a half-life typical ofhuman IgG4 antibodies.

To assess the effect of anti-LILRB2 on neutrophil populations, a CBCassay was conducted in cynomolgus monkeys pre-study and following dosingof anti-LILRB2 antibodies. As shown in FIGS. 26A and 26B, peripheralblood neutrophils remained within normal range and displayed anonsignificant downward trend.

Example 16. Pirb Knockout Mice Experiment

Eight- to twelve-week old Pirb homozygous knockout mice or wild typelittermate controls were inoculated subcutaneously with B16.SIY (1×10⁶),LLC (2×10⁶), or MC38 (5×10⁵) tumor cells. Once palpable tumors werefelt, mice were monitored and tumor measurements recorded at least twiceweekly until tumors exceeded 2,000 mm³ or mice had a body weightdecrease of over 20%. Some mice were sacrificed early for analysis ofthe tumor-infiltrating cells. All experiments were conducted inaccordance with institutional guidelines for animal care and use.

Tumors were detected in all wild type mice and in line with historicalgrowth kinetics at Jounce Therapeutics. While no significant differencewas observed in the growth of B16.SIY or LLC tumors between wild typeand Pirb knockout mice (FIGS. 27A, 27B, 28A, and 28B), a significantdecrease in MC38 tumor growth was found in Pirb knockout as compared towild type mice (FIGS. 29A and 29B). Analysis of the MC38tumor-infiltrating cells was consistent with a phenotype oftumor-associated macrophages that exhibited less immunosuppressivecharacteristics (lower IL-4R) and increased antigen presentationcapacity (higher MHC class II) in Pirb knockout mice as compared to wildtype controls (data not shown).

Example 17. Alanine Scan Analysis

The heavy chain and light chain variable regions of J-19.h1 are setforth in FIG. 30. The complementarity-determining region (CDR) of theheavy and light chains of J-19.h1 as defined by the Kabat CDR definitionare indicated in FIG. 30 by underlining. Key binding residues of J-19.h1necessary for binding the target, LILRB2, were identified by mutatingindividual residues in the CDRs. The scan was conducted by mutating 34heavy chain and 18 light chain residues in the CDRs individually toalanine. Mutations to CDRL2 were not included as this region of J-19.h1was not determined to contribute to LILRB2 binding. A full mutation ofJ-19.h1 to the germline CDRL2 sequence did not change binding affinityfor the target.

All variants were produced transiently in a Chinese Hamster Ovarian cellline and purified using into citrate buffer on an automated liquidhandler. Affinities were determined using a ForteBio Octet Red96.J-19.h1 variants were normalized to 10 ug/mL and loaded onto anti-humancapture sensors. The loaded sensors were then soaked in kinetics bufferto establish a baseline and dipped into wells containing LILRB2 at twoconcentrations of 50 nM and 5 nM, followed by a dissociation step intobuffer. Kinetics data was calculated using the ForteBio data analysissoftware.

Resulting affinities of J-19.h1 variants to LILRB2 showed key residues,highlighted grey, are critical to LILRB2 binding. Mutation to alanineresulted in affinities that were 5-fold less than the control orresulted in complete abrogation of binding. Residues with a dottedunderline had affinities between 2 and 5-fold of the control. All otherresidues in the CDR maintained similar affinity to LILRB2 when mutatedto alanine.

Example 18. qPCR Primers

Commercially available qPCR assays for LILRB2 from both AppliedBiosystems (TaqMan) and Biorad (PrimePCR) did not show specificity fortheir target when run on cDNA derived from the RNA of LILR family memberover-expressing cell lines. The LILR family members have high homologywith each other and primer sets therefore can produce off target effectsduring PCR.

The following primer sets were tested on the over-expressing cell linesand one primer set showed specificity under certain PCR conditions. ThecDNA libraries were generated using both the BioRad iScript ReverseTranscription Supermix and the Fluidigm Reverse Transcription mastermix. The primer sets, when not accompanied with a probe, were run usingthe BioRad SsoFast EvaGreen Supermix with Low ROX master mix. The primersets with probes were run using the Applied Biosystems TaqMan FastAdvanced Master Mix. The primers were ordered from IDT as oligos and theprobes were labeled with FAM.

TABLE 8 Primer and probe sequences Forward Primer Reverse PrimerProbe (if any) Set CACACAGCTCAAC TGGGTAGGCTCC 1 CTGGACA TGTCATCA SetAGCTCAACCTGGA CTTGGGGATGGT 2 CAGCAC CCCTGTCT Set ACACACAGCTCAACAGGCAGACTCA 3 CCTGGAC GATCAGCA Set CACACAGCTCAAC CTGCAGGCAGAC 4 CTGGACATCAGATCA Set CCTGCATTTCTCC CTGTCCAGGTTG 5 TCTGTGC AGCTGTGT SetTCGCACAGGTGCT GCACTGAGAGTG 6 ATGGTTA ATGGCTTCTTA Set AGTAGAAGGAGACTCCCAAAGTTCC AGCCTGGACCCC 7 TCAGGACTG CAGCATC TAACAAAGACC SetTTCCACACTTTCC GGGAATTCAGCC TGCCCCACTCCGTC 8 TTCTGACC TGGTACTTAGTAAGATCAATACA Set CGTCACCCTCAGT TCCGTGTAATCC CCTTGAAGCCCAGG 9 TGTCAGAAGATGCTG AGTACCGTCTA Set CCTACTTCCCTGC CAGGCAGACTCA AGCTCAACCTGGAC 10ATTTCTCC GATCAGC GGCACA Set TTCTTCCCCTACT CTTCAAGGCTCC 11 TCCCTGCATTTCCCTGACAAC Set GAAGTCAACTTTT CAAGGCTCCCCT 12 CTTCCCCTAC GACAACT SetCACACAGCTCAAC AGACTCAGCCCG 13 CTGGACA AGACAGAT Set GTCAACTTTTCTAAGGCTCCCCTG 14 TCCCCTACTTC ACAACTG Set AAGAAGCCATC GTAGGAGCGGCT 15ACTCTCAGTGC CACAGGThe forward primers for sets 1-15 are SEQ ID NOs: 135-149; the reverseprimers for sets 1-15 are SEQ ID NOs: 150-164; the probes for sets 7-10are SEQ ID NOs: 165-168; each in order as set forth in Table 8.

Set 9 listed above proved to be specific for LILRB2 when assayed acrossthe numerous LILR family member over-expressing cell lines when run as aTaqMan assay (both Applied Biosystems master mix and IDT GE master mix),as well as a primer set EvaGreen assay (BioRad Master mix). Thefollowing PCR cycling conditions were followed: Cycle 1: 95° C. for 60seconds; Cycle 2: 96° C. for 5 seconds; Cycle 3: 65° C. for 20 seconds;Repeat cycles 2-3 for 40 cycles total; Melting curve for EvaGreenchemistry from 60-95° C. Primer and probe sequences: Forward:CGTCACCCTCAGTTGTCAG (SEQ ID NO: 143); Reverse: TCCGTGTAATCCAAGATGCTG(SEQ ID NO: 158); Probe: CCTTGAAGCCCAGGAGTACCGTCTA (SEQ ID NO: 167).

The addition of up to 5 nucleotides to either end of the primers shouldnot affect PCR specificity per the UCSC In-Silico PCR tool (bold iswet-validated primer set).https://genome.ucsc.edu/cgi-bin/hgPcr?hgsid=693240227_npi8w0U7mF4EWHuVaOYA4nIqdtsZAGTCC-CGTCACCCTCAGTTGTCAG-GGGAG (SEQ ID NO: 169);TCGTA-TCCGTGTAATCCAAGATGCTG-ATTTT (SEQ ID NO: 170).

Example 19. PD Signature Score Analysis of Fresh Tumor Samples

Fresh human tumor samples were obtained post-surgery. A section of eachtumor was cut and fixed for IHC. 300 μM slices of remaining tumor wereplaced in a 6-well plate. Treatments were added into the medium andplates were incubated at 37° C. Tumor slices were stored in RNAlaterafter incubation. Each slice was treated with 10 μg/mL of drug for 24hours; in instances where samples were treated with more than one drug,10 μg/mL of each drug was used.

Tumor slices were lysed using Qiagen's TissueLyser processor and FFPEsamples were deparaffinized. RNA was extracted from FFPE and fresh tumorsamples, quantified using Quibit, and QC′d using AATI's FragmentAnalyzer. If sufficient RNA was extracted from the sample, geneexpression was performed using NanoString nCounter using the HumanImmunology V2 panel as well as a custom macrophage-specific spike-in.Gene expression was normalized to the expression of housekeeping genes,then noise thresholding was performed using the data from negativeprobes and data was transformed to the log 2 space. This data willhenceforth be referred to as “normalized gene expression.” Normalizedgene expression data was then further normalized to the average datafrom the palivizumab treated samples from the same patient. This datawill henceforth be referred to as “palivizumab-normalized geneexpression.”

Pharmacodynamic (PD) signature scores were calculated for each sample.Monoculture signatures are derived from the mean log 2 (fold changecompared to palivizumab-treated samples) in gene expression acrossmonocyte-derived macrophages from 4 donors in response to anti-LILRB2ligand-blocking drugs in the absence of LPS after 4 hours. “Monoculturesignature scores” were calculated for each treated histoculture sampleby projecting the palivizumab-normalized gene expression onto the vectordefined by the monoculture signature. “IFNγ signature scores” werecalculated by averaging the palivizumab-normalized gene expression ofthe 6 genes identified by Hirsch et al. (32^(nd) Annual Meeting andPre-Conference Programs of the Society for Immunotherapy of Cancer (SITC2017): Part One, P39; J. Immunother. Cancer 5 (Suppl. 2):86, 2017)displaying modulated expression in response to anti-PD1 treatment.“Keytruda signature scores” were calculated by averaging thepalivizumab-normalized gene expression of the 18 genes identified byAyers et al. (J. Clin. Invest. 127:2930-2940, 2017) as being predictiveof clinical response to pembrolizumab.

To evaluate noise in the system and determine response cutoffs, 173tumor slices (samples) from 80 kidney, lung, and head and neck tumorswere treated with the control antibody palivizumab for 24 hours. Atleast two samples from each tumor were treated with palivizumab. Thenoise threshold for each PD response signature was defined as the 95thpercentile of the distribution of the signature scores across the 173samples. Tumors are classified as “responders” to a particular drug ifthe average PD signature score across all samples from that tumortreated with that drug is greater than the noise threshold for thatsignature.

Baseline (untreated) samples from most tumors were characterized. Celltype specific signatures were calculated by averaging the normalizedgene expression of genes that are associated with particular cell types.The Keytruda signature was calculated for each baseline sample byaveraging the normalized gene expression of the 18 genes identified byAyers et al. (supra) as being predictive of clinical response topembrolizumab.

Results of the experiments described above are shown in FIGS. 31-34.

FIG. 31 is a histogram of the IFNγ PD response scores from 173 tumorsamples from 80 tumors treated with palivizumab for 24 hours. In eachtumor, at least two samples were treated with palivizumab. The noisethreshold for the signature is defined as the 95th percentile of thedistribution. For the IFNγ signature, the noise threshold is 0.43.

FIG. 32 is a Venn diagram and chart describing the PD response rates toJ-19.h1 across 3 indications: renal cell carcinoma, head and neckcancer, and lung cancer. Tumors are classified as “responders” if theaverage PD response score across all J-19.H1 treated slices for thattumor is greater than the noise threshold. As noted above, the noisethreshold for each PD signature is defined as the 95th percentile of thedistribution of PD response scores of palivizumab treated samples acrosstumors with more than one palivizumab treated sample. In histoculture,J-19.H1 induces different PD responses across indications, suggestingmultiple mechanisms of action: the monoculture signature indicatesmacrophage polarization; the IFN gamma signature suggests similarresponse to checkpoint inhibitors; the Keytruda signature is a sign oftumor priming for response to checkpoint inhibitors.

FIG. 33 is a series of graphs showing Keytruda signature scorescalculated for untreated samples, based on normalized gene expression(raw gene expression is normalized to housekeeping genes and negativecontrol probes, then log 2 transformed). Tumors are classified as IFNγPD responders if the average response of all samples in that tumortreated with J-19.H1 is greater than the noise threshold of the IFNγsignature. Each dot represents the Keytruda signature score of anuntreated tumor sample. Dotted lines show the average baseline Keytrudasignature score for the samples profiled in each indication. Thebaseline Keytruda signature score is a necessary but insufficientcondition for IFN gamma PD response to pembrolizumab in histoculture,which is consistent with clinical observations reported by others, thussuggesting the relevance of the histoculture model to clinical outcomes.

FIG. 34, left panel, is a table showing average IFNγ PD signature scorescalculated for 18 head and neck tumors in response to J-19.h1,pembrolizumab, or J-19.H1 combined with pembrolizumab. Cells highlightedin grey indicate tumors for which the response to treatment is greaterthan the noise threshold. Rows with an asterisk beside them denotetumors for which J-19.h1 potentiates response; i.e., the IFNγ PDsignature score in response to J-19.H1+pembrolizumab is greater than orequal to the score in response to pembrolizumab alone+0.43 (the noisethreshold for the IFNγ PD signature). Tumors that have potentiatedresponse with J-19.H1 are referred to as having a “combo effect” in theright panel. FIG. 34, right panel, is a graph showing a comparison ofthe indication-normalized tumor macrophage content of tumors prior totreatment. The graph shows the baseline macrophage content of tumorsthat display a combination effect in response to J-19.H1+pembrolizumabcompared to those that do not show any potentiation due to J-19.H1. Thecomparison is made for tumors across 3 indications: renal cellcarcinoma, lung cancer and head, and neck cancer. Tumor macrophagecontent is calculated by averaging the expression of genes associatedwith macrophages in untreated samples and is normalized within eachindication. The IFN gamma PD response to pembrolizumab in histocultureis potentiated by J-19.H1 in samples enriched with macrophages.

Example 20. Antibody Mutants

The heavy chain variable region of J-19.h1 is set forth below:

                          (SEQ ID NO: 53)QITLKESGPTLVKPTQTLTLTCTFSGFSLNTYAMGVSWI

LVTVSS

CDRH1, CDRH2, and CDRH3 are indicated by underlining, in order. R*denotes a residue in CDRH3 that, when mutate to alanine (J-19.h5),measured higher affinity for LILRB2 compared to J-19.h1. Thismeasurement was determined using a ForteBio Octet Red96. The variant wasnormalized to 10 ug/mL and loaded onto an anti-human capture sensor. Theloaded sensor was then soaked in kinetics buffer to establish a baselineand dipped into wells containing LILRB2 at two concentrations of 50 nMand 5 nM, followed by a dissociation step into buffer. Kinetics data wascalculated using the ForteBio data analysis software. Results showedJ-19.h5 having a two-fold higher affinity for LILRB2 compared to theunmutated version of J-19.h1.

Additional variants were made of J-19.h1, in which the arginine wasmutated to aspartate (J-19.h6) and glutamate (J-19.h7). All variantswere produced transiently using a Chinese Hamster Ovarian cell line andpurified using citrate buffer on an automated liquid handler. Affinityto LILRB2 was measured exactly as it was previously to J-19.h5. The dataindicated that these two variants have even greater affinity to LILRB2compared to J-19.h1 and J-19.h5.

Affinities for the four antibodies (J-19.h1, J-19.h5, J-19.h6, andJ-19.h7) were confirmed through surface plasmon resonance (SPR) using aSierraSensor Mass-2. The antibodies were captured using an anti-human Fcchip at a concentration of 2 ug/mL. LILRB2 was flowed over the capturedantibodies at seven concentrations (65, 21.67, 7.22, 2.41, 0.802, 0.267,0.089 nM). J-19.h5 and J-19.h6 were run in triplicates, while J-19.h7and J-19.h1 were run in duplicates. Below is a table describing theassociation, dissociation, and Kd of the four antibodies.

TABLE 9 Kinetic Measurements of J-19.h1 and Higher Affinity Mutants Folddifference Repli- over Analyte Ligand Ka (1/Ms) Kd (1/s) KD (M) cates3-19.h1 LILRB2 3-19.h5 8.76E+05 1.15E−03 1.31E−09 3 1.91 3-19.h68.25E+05 5.92E−04 7.18E−10 3 3.47 3-19.h7 8.65E+05 7.88E−04 9.12E−10 22.73 3-19.h1 7.05E+05 1.76E−03 2.49E−09 2 1  

TABLE 10 Table of Sequences SEQ ID NO: Description Sequence 1J-11.h heavy chain QVQLQQSGAELMKPGASVKLSCKATGYILTGYWIEWVKQRPGHGLEWIGEILPGSGSTNYNENFKGKATFTADTSSNTAYMQLSSLTTEDSAIYYCARAVLGYFDYWGQGTTLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 2 J-11.h light chainDIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDYTLTISSVQAEDLALYYCHQHYSTYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC 3 J-11.h V_(H)QVQLQQSGAELMKPGASVKLSCKATGYILTGYWIEWVKQRPGHGLEWIGEILPGSGSTNYNENFKGKATFTADTSSNTAYMQLSSLTTEDSAIYYCARAVLGYFDYWGQGTTLTVSS 4 J-11.h V_(L)DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDYTLTISSVQAEDL ALYYCHQHYSTYTFGGGTKLEIK 5J-11.h CDR-H1 GYWIE 6 J-11.h CDR-H2 EILPGSGSTNYNENFKG 7 J-11.h CDR-H3AVLGYFDY 8 J-11.h CDR-L1 KASQDVSTAVA 9 J-11.h CDR-L2 WASTRHT 10J-11.h CDR-L3 HQHYSTYT 11 J-19.h heavy chainQVTLKESGPGILQPSHTLSLTCSFSGFSLNTYAMGVSWIRQPSGKGLEWLASIWWNGNKYNNPSLKSRLTVSKDTSNNQAFLKVTSVDTADTATYYCAHSRIIRFTDYVMDAWGQGASVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 12 J-19.h light chainDIQMTQSPASLSTFLGEPVTIECRASEDIYNDLAWYQQKPGKSPQLLIYNANSLHTGVPSRFSGSGSGTQYSLKINSLQSEDVASYFCQQYYDYPLTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 13 J-19.h V_(H)QVTLKESGPGILQPSHTLSLTCSFSGFSLNTYAMGVSWIRQPSGKGLEWLASIWWNGNKYNNPSLKSRLTVSKDTSNNQAFLKVTSVDTADTATYYCAHSRIIRFTDYVMDAWGQGASVTVSS 14 J-19.h V_(L)DIQMTQSPASLSTFLGEPVTIECRASEDIYNDLAWYQQKPGKSPQLLIYNANSLHTGVPSRFSGSGSGTQYSLKINSLQSEDVA SYFCQQYYDYPLTFGSGTKLEIK 15J-19.h CDR-H1 TYAMGVS 16 J-19.h CDR-H2 SIWWNGNKYNNPSLKS 17 J-19.h CDR-H3SRIIRFTDYVMDA 18 J-19.h CDR-L1 RASEDIYNDLA 19 J-19.h CDR-L2 NANSLHT 20J-19.h CDR-L3 QQYYDYPLT 21 J-17.h heavy chainQIQLVQSGPELKKPGETVKISCKASGYTFTTYGLSWVKQTPGKGLKWMGWINTYSGVPTYTDDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARPYDFDQVGFAYWGQGTLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 22 J-17.h light chainSIVMTQTPKFLLVSAGDRVSITCKASQTVSSDVAWYQQKAGQSPKLLIYYASNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPFTFGGGSKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC 23 J-17.h V_(H)QIQLVQSGPELKKPGETVKISCKASGYTFTTYGLSWVKQTPGKGLKWMGWINTYSGVPTYTDDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARPYDFDQVGFAYWGQGTLVTVSA 24 J-17.h V_(L)SIVMTQTPKFLLVSAGDRVSITCKASQTVSSDVAWYQQKAGQSPKLLIYYASNRYTGVPDRFTGSGYGTDFTFTISTVQAEDL AVYFCQQDYSSPFTFGGGSKLEIK 25J-17.h CDR-H1 TYGLS 26 J-17.h CDR-H2 WINTYSGVPTYTDDFKG 27 J-17.h CDR-H3PYDFDQVGFAY 28 J-17.h CDR-L1 KASQTVSSDVA 29 J-17.h CDR-L2 YASNRYT 30J-17.h CDR-L3 QQDYSSPFT 31 J-04 heavy chainQVQLQQSGAELVRPGASVTLSCKASGYTFADYEIHWVKQTPVHGLEWIGAIDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRYYDYDDAMDYWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 32 J-04 light chainQIVLTQSPAIMSASPGEKVTMTCSASSSVSFMHWYQQKSGTSPKRWIYGTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWNGNPFTFGSGTKLETKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC 33 J-04 V_(H)QVQLQQSGAELVRPGASVTLSCKASGYTFADYEIHWVKQTPVHGLEWIGAIDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRYYDYDDAMDYWGQGTSVTVSS 34 J-04 V_(L)QIVLTQSPAIMSASPGEKVTMTCSASSSVSFMHWYQQKSGTSPKRWIYGTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAA TYYCQQWNGNPFTFGSGTKLETK 35J-04 CDR-H1 DYEIH 36 J-04 CDR-H2 AIDPETGGTAYNQKFKG 37 J-04 CDR-H3YYDYDDAMDY 38 J-04 CDR-L1 SASSSVSFMH 39 J-04 CDR-L2 GTSKLAS 40J-04 CDR-L3 QQWNGNPFT 41 J-03 heavy chainQVQLQQSGAELVRPGASVTLSCKASGYKFTDYEMHWVKQTPVHGLEWIGAIDPETNGTAYNKKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRGDYDFSAWFAYWGQGTLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 42 J-03 light chainDIVLTQSPASLAVSLGQRATISCRASESVDNYDISFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVETDDVATYYCQQSNKDPRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC 43 J-03 V_(H)QVQLQQSGAELVRPGASVTLSCKASGYKFTDYEMHWVKQTPVHGLEWIGAIDPETNGTAYNKKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRGDYDFSAWFAYWGQGTLVTVSA 44 J-03 V_(L)DIVLTQSPASLAVSLGQRATISCRASESVDNYDISFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVETD DVATYYCQQSNKDPRTFGGGTKLEIK45 J-03 CDR-H1 DYEMH 46 J-03 CDR-H2 AIDPETNGTAYNKKFKG 47 J-03 CDR-H3GDYDFSAWFAY 48 J-03 CDR-L1 RASESVDNYDISFMH 49 J-03 CDR-L2 RASNLES 50J-03 CDR-L3 QQSNKDPRT 51 J-19.h1 heavy chainQITLKESGPTLVKPTQTLTLTCTFSGFSLNTYAMGVSWIRQPPGKALEWLASIWWNGNKYNNPSLKSRLTVTKDTSKNQVVLTMTNMDPVDTATYYCAHSRIIRFTDYVMDAWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 52 J-19.h1 light chainDIQMTQSPSSLSTSVGDRVTITCRASEDIYNDLAWYQQKPGKAPKLLIYNANSLHTGVASRFSGSGSGTDFTFTISSLQPEDVATYFCQQYYDYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 53 J-19.h1 V_(H)QITLKESGPTLVKPTQTLTLTCTFSGFSLNTYAMGVSWIRQPPGKALEWLASIWWNGNKYNNPSLKSRLTVTKDTSKNQVVLTMTNMDPVDTATYYCAHSRIIRFTDYVMDAWGQGTLVTVSS 54 J-19.h1 V_(L)DIQMTQSPSSLSTSVGDRVTITCRASEDIYNDLAWYQQKPGKAPKLLIYNANSLHTGVASRFSGSGSGTDFTFTISSLQPEDVAT YFCQQYYDYPLTFGQGTKLEIK 55J-19.h1 CDR-H1 TYAMGVS 56 J-19.h1 CDR-H2 SIWWNGNKYNNPSLKS 57J-19.h1 CDR-H3 SRIIRFTDYVMDA 58 J-19.h1 CDR-L1 RASEDIYNDLA 59J-19.h1 CDR-L2 NANSLHT 60 J-19.h1 CDR-L3 QQYYDYPLT 61J-19.h2 heavy chain QITLKESGPTLVKPTQTLTLTCTFSGFSLNTYAMGVSWIRQPPGKALEWLASIWWNGNKYNNPSLKSRLTVTKDTSKNQVVLTMTNMDPVDTATYYCAHSRIIRFTDYVMDAWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 62 J-19.h2 light chainDIVMTQSPSSLSASVGDTVTITCRASEDIYNDLAWYQQKPGKAPQLLLYNANSLHTGVPSRFSGSGSGTDYTLTISTLQPEDFATYYCQQYYDYPLTFGPGTKVHIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC 63 J-19.h2 V_(H)QITLKESGPTLVKPTQTLTLTCTFSGFSLNTYAMGVSWIRQPPGKALEWLASIWWNGNKYNNPSLKSRLTVTKDTSKNQVVLTMTNMDPVDTATYYCAHSRIIRFTDYVMDAWGQGTLVTVSS 64 J-19.h2 V_(L)DIVMTQSPSSLSASVGDTVTITCRASEDIYNDLAWYQQKPGKAPQLLLYNANSLHTGVPSRFSGSGSGTDYTLTISTLQPEDFA TYYCQQYYDYPLTFGPGTKVHIK 65J-19.h2 CDR-H1 TYAMGVS 66 J-19.h2 CDR-H2 SIWWNGNKYNNPSLKS 67J-19.h2 CDR-H3 SRIIRFTDYVMDA 68 J-19.h2 CDR-L1 RASEDIYNDLA 69J-19.h2 CDR-L2 NANSLHT 70 J-19.h2 CDR-L3 QQYYDYPLT 71J-19.h3 heavy chain QVTLKESGPSLVKPTQTLTLTCTFSGFSLNTYAMGVSWIRQPPGKALEWLASIWWNGNKYNNPSLKSRLTITKDTSKNQVVLKVTNMDPADTATYYCAHSRIIRFTDYVMDAWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 72 J-19.h3 light chainDIVMTQSPSSLSASVGDTVTITCRASEDIYNDLAWYQQKPGKAPQLLLYNANSLHTGVPSRFSGSGSGTDYTLTISTLQPEDFATYYCQQYYDYPLTFGPGTKVHIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC 73 J-19.h3 V_(H)QVTLKESGPSLVKPTQTLTLTCTFSGFSLNTYAMGVSWIRQPPGKALEWLASIWWNGNKYNNPSLKSRLTITKDTSKNQVVLKVTNMDPADTATYYCAHSRIIRFTDYVMDAWGQGTTVTVSS 74 J-19.h3 V_(L)DIVMTQSPSSLSASVGDTVTITCRASEDIYNDLAWYQQKPGKAPQLLLYNANSLHTGVPSRFSGSGSGTDYTLTISTLQPEDFA TYYCQQYYDYPLTFGPGTKVHIK 75J-19.h3 CDR-H1 TYAMGVS 76 J-19.h3 CDR-H2 SIWWNGNKYNNPSLKS 77J-19.h3 CDR-H3 SRIIRFTDYVMDA 78 J-19.h3 CDR-L1 RASEDIYNDLA 79J-19.h3 CDR-L2 NANSLHT 80 J-19.h3 CDR-L3 QQYYDYPLT 81J-19.h4 heavy chain QVTLKESGPALVKPTHTLTLTCTFSGFSLNTYAMGVSWIRQPPGKALEWLASIWWNGNKYNNPSLKSRLTISKDTSKNQVVLTMTNMDPEDTATFYCAHSRIIRFTDYVMDAWGRGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 82 J-19.h4 light chainDIVMTQSPSSLSASVGDTVTITCRASEDIYNDLAWYQQKPGKAPQLLLYNANSLHTGVPSRFSGSGSGTDYTLTISTLQPEDFATYYCQQYYDYPLTFGPGTKVHIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC 83 J-19.h4 V_(H)QVTLKESGPALVKPTHTLTLTCTFSGFSLNTYAMGVSWIRQPPGKALEWLASIWWNGNKYNNPSLKSRLTISKDTSKNQVVLTMTNMDPEDTATFYCAHSRIIRFTDYVMDAWGRGTTVTVSS 84 J-19.h4 V_(L)DIVMTQSPSSLSASVGDTVTITCRASEDIYNDLAWYQQKPGKAPQLLLYNANSLHTGVPSRFSGSGSGTDYTLTISTLQPEDFA TYYCQQYYDYPLTFGPGTKVHIK 85J-19.h4 CDR-H1 TYAMGVS 86 J-19.h4 CDR-H2 SIWWNGNKYNNPSLKS 87J-19.h4 CDR-H3 SRIIRFTDYVMDA 88 J-19.h4 CDR-L1 RASEDIYNDLA 89J-19.h4 CDR-L2 NANSLHT 90 J-19.h4 CDR-L3 QQYYDYPLT 91J-19.h5 heavy chain QITLKESGPTLVKPTQTLTLTCTFSGFSLNTYAMGVSWIRQPPGKALEWLASIWWNGNKYNNPSLKSRLTVTKDTSKNQVVLTMTNMDPVDTATYYCAHSRIIAFTDYVMDAWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 92 J-19.h5 light chainDIQMTQSPSSLSTSVGDRVTITCRASEDIYNDLAWYQQKPGKAPKLLIYNANSLHTGVASRFSGSGSGTDFTFTISSLQPEDVATYFCQQYYDYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 93 J-19.h5 V_(H)QITLKESGPTLVKPTQTLTLTCTFSGFSLNTYAMGVSWIRQPPGKALEWLASIWWNGNKYNNPSLKSRLTVTKDTSKNQVVLTMTNMDPVDTATYYCAHSRIIAFTDYVMDAWGQGTLVTVSS 94 J-19.h5 V_(L)DIQMTQSPSSLSTSVGDRVTITCRASEDIYNDLAWYQQKPGKAPKLLIYNANSLHTGVASRFSGSGSGTDFTFTISSLQPEDVAT YFCQQYYDYPLTFGQGTKLEIK 95J-19.h5 CDR-H1 TYAMGVS 96 J-19.h5 CDR-H2 SIWWNGNKYNNPSLKS 97J-19.h5 CDR-H3 SRIIAFTDYVMDA 98 J-19.h5 CDR-L1 RASEDIYNDLA 99J-19.h5 CDR-L2 NANSLHT 100 J-19.h5 CDR-L3 QQYYDYPLT 101J-19.h6 heavy chain QITLKESGPTLVKPTQTLTLTCTFSGFSLNTYAMGVSWIRQPPGKALEWLASIWWNGNKYNNPSLKSRLTVTKDTSKNQVVLTMTNMDPVDTATYYCAHSRIIDFTDYVMDAWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 102 J-19.h6 light chainDIQMTQSPSSLSTSVGDRVTITCRASEDIYNDLAWYQQKPGKAPKLLIYNANSLHTGVASRFSGSGSGTDFTFTISSLQPEDVATYFCQQYYDYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 103 J-19.h6 V_(H)QITLKESGPTLVKPTQTLTLTCTFSGFSLNTYAMGVSWIRQPPGKALEWLASIWWNGNKYNNPSLKSRLTVTKDTSKNQVVLTMTNMDPVDTATYYCAHSRIIDFTDYVMDAWGQGTLVTVSS 104 J-19.h6 V_(L)DIQMTQSPSSLSTSVGDRVTITCRASEDIYNDLAWYQQKPGKAPKLLIYNANSLHTGVASRFSGSGSGTDFTFTISSLQPEDVAT YFCQQYYDYPLTFGQGTKLEIK 105J-19.h6 CDR-H1 TYAMGVS 106 J-19.h6 CDR-H2 SIWWNGNKYNNPSLKS 107J-19.h6 CDR-H3 SRIIDFTDYVMDA 108 J-19.h6 CDR-L1 RASEDIYNDLA 109J-19.h6 CDR-L2 NANSLHT 110 J-19.h6 CDR-L3 QQYYDYPLT 111J-19.h7 heavy chain QITLKESGPTLVKPTQTLTLTCTFSGFSLNTYAMGVSWIRQPPGKALEWLASIWWNGNKYNNPSLKSRLTVTKDTSKNQVVLTMTNMDPVDTATYYCAHSRIIEFTDYVMDAWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 112 J-19.h7 light chainDIQMTQSPSSLSTSVGDRVTITCRASEDIYNDLAWYQQKPGKAPKLLIYNANSLHTGVASRFSGSGSGTDFTFTISSLQPEDVATYFCQQYYDYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 113 J-19.h7 V_(H)QITLKESGPTLVKPTQTLTLTCTFSGFSLNTYAMGVSWIRQPPGKALEWLASIWWNGNKYNNPSLKSRLTVTKDTSKNQVVLTMTNMDPVDTATYYCAHSRIIEFTDYVMDAWGQGTLVTVSS 114 J-19.h7 V_(L)DIQMTQSPSSLSTSVGDRVTITCRASEDIYNDLAWYQQKPGKAPKLLIYNANSLHTGVASRFSGSGSGTDFTFTISSLQPEDVAT YFCQQYYDYPLTFGQGTKLEIK 115J-19.h7 CDR-H1 TYAMGVS 116 J-19.h7 CDR-H2 SIWWNGNKYNNPSLKS 117J-19.h7 CDR-H3 SRIIEFTDYVMDA 118 J-19.h7 CDR-L1 RASEDIYNDLA 119J-19.h7 CDR-L2 NANSLHT 120 J-19.h7 CDR-L3 QQYYDYPLT SEQ ID NO: ProteinAcc. No. Sequence 121 LILRA1 O75019MTPIVTVLICLRLSLGPRTHVQAGTLPKPTLWAEPGSVITQGSPVTLWCQGILETQEYRLYREKKTAPWITRIPQEIVKKGQFPIPSITWEHTGRYRCFYGSHTAGWSEPSDPLELVVTGAYIKPTLSALPSPVVTSGGNVTLHCVSQVAFGSFILCKEGEDEHPQCLNSQPRTHGWSRAIFSVGPVSPSRRWSYRCYAYDSNSPHVWSLPSDLLELLVLGVSKKPSLSVQPGPIVAPGESLTLQCVSDVSYDRFVLYKEGERDFLQLPGPQPQAGLSQANFTLGPVSRSYGGQYRCSGAYNLSSEWSAPSDPLDILIAGQFRGRPFISVHPGPTVASGENVTLLCQSWGPFHTFLLTKAGAADAPLRLRSIHEYPKYQAEFPMSPVTSAHSGTYRCYGSLSSNPYLLSHPSDSLELMVSGAAETLSPPQNKSDSKAGAANTLSPSQNKTASHPQDYTVENLIRMGIAGLVLVVLGILLFEAQHSQRSL 122 LILRA2 Q8N149MTPILTVLICLGLSLGPRTHVQAGHLPKPTLWAEPGSVIIQGSPVTLRCQGSLQAEEYHLYRENKSASWVRRIQEPGKNGQFPIPSITWEHAGRYHCQYYSHNHSSEYSDPLELVVTGAYSKPTLSALPSPVVTLGGNVTLQCVSQVAFDGFILCKEGEDEHPQRLNSHSHARGWSWAIFSVGPVSPSRRWSYRCYAYDSNSPYVWSLPSDLLELLVPGVSKKPSLSVQPGPMVAPGESLTLQCVSDVGYDRFVLYKEGERDFLQRPGWQPQAGLSQANFTLGPVSPSHGGQYRCYSAHNLSSEWSAPSDPLDILITGQFYDRPSLSVQPVPTVAPGKNVTLLCQSRGQFHTFLLTKEGAGHPPLHLRSEHQAQQNQAEFRMGPVTSAHVGTYRCYSSLSSNPYLLSLPSDPLELVVSEAAETLSPSQNKTDSTTTSLGQHPQDYTVENLIRMGVAGLVLVVLGILLFEAQHSQRSLQDAAGR 123 LILRA3 Q8N6C8MTPILTVLICLGLSLDPRTHVQAGPLPKPTLWAEPGSVITQGSPVTLRCQGSLETQEYHLYREKKTALWITRIPQELVKKGQFPILSITWEHAGRYCCIYGSHTAGLSESSDPLELVVTGAYSKPTLSALPSPVVTSGGNVTIQCDSQVAFDGFILCKEGEDENPQCLNSHSHARGSSRAIFSVGPVSPSRRWSYRCYGYDSRAPYVWSLPSDLLGLLVPGVSKKPSLSVQPGPVVAPGEKLTFQCGSDAGYDRFVLYKEWGRDFLQRPGRQPQAGLSQANFTLGPVSRSYGGQYTCSGAYNLSSEWSAPSDPLDILITGQIRARPFLSVRPGPTVASGENVTLLCQSQGGMHTFLLTKEGAADSPLRLKSKRQSHKYQAEFPMSPVTSAHAGTYRCYGSLSSNPYLLTHPSDPL ELVVSGAAETLSPPQNKSDSKAGE 124LILRA4 P59901 MTLILTSLLFFGLSLGPRTRVQAENLPKPILWAEPGPVITWHNPVTIWCQGTLEAQGYRLDKEGNSMSRHILKTLESENKVKLSIPSMMWEHAGRYHCYYQSPAGWSEPSDPLELVVTAYSRPTLSALPSPVVTSGVNVTLRCASRLGLGRFTLIEEGDHRLSWTLNSHQHNHGKFQALFPMGPLTFSNRGTFRCYGYENNTPYVWSEPSDPLQLLVSGVSRKPSLLTLQGPVVTPGENLTLQCGSDVGYIRYTLYKEGADGLPQRPGRQPQAGLSQANFTLSPVSRSYGGQYRCYGAHNVSSEWSAPSDPLDILIAGQISDRPSLSVQPGPTVTSGEKVTLLCQSWDPMFTFLLTKEGAAHPPLRLRSMYGAHKYQAEFPMSPVTSAHAGTYRCYGSRSSNPYLLSHPSEPLELVVSGATETLNPAQKKSDSKTAPHLQDYTVENLIRMGVAGLVLLFLGILLFEAQHSQRSPPRCSQEANSRKDNAPFRVVEP WEQI 125 LILRA5 A6NI73MAPWSHPSAQLQPVGGDAVSPALMVLLCLGLSLGPRTHVQAGNLSKATLWAEPGSVISRGNSVTIRCQGTLEAQEYRLVKEGSPEPWDTQNPLEPKNKARFSIPSMTEHHAGRYRCYYYSPAGWSEPSDPLELVVTGFYNKPTLSALPSPVVTSGENVTLQCGSRLRFDRFILTEEGDHKLSVVTLDSQLTPSGQFQALFPVGPVTPSHRWMLRCYGSRRHILQVWSEPSDLLEIPVSGAADNLSPSQNKSDSGTASHLQDYAVENLIRMGMAGLILVVLGILIFQDWH SQRSPQAAAGR 126 LILRA6 Q6PI73MTPALTALLCLGLSLGPRTRVQAGPFPKPTLWAEPGSVISWGSPVTIWCQGSLEAQEYQLDKEGSPEPLDRNNPLEPKNKARFSIPSMTQHHAGRYRCHYYSSAGWSEPSDPLELVMTGFYNKPTLSALPSPVVASGGNMTLRCGSQKGYHHFVLMKEGEHQLPRTLDSQQLHSGGFQALFPVGPVTPSHRWRFTCYYYYTNTPRVWSHPSDPLEILPSGVSRKPSLLTLQGPVLAPGQSLTLQCGSDVGYDRFVLYKEGERDFLQRPGQQPQAGLSQANFTLGPVSPSHGGQYRCYGAHNLSSEWSAPSDPLNILMAGQIYDTVSLSAQPGPTVASGENVTLLCQSRGYFDTFLLTKEGAAHPPLRLRSMYGAHKYQAEFPMSPVTSAHAGTYRCYGSYSSNPHLLSFPSEPLELMVSGHSGGSSLPPTGPPSTPASHAKDYTVENLIRMGMAGLVLVFLGILLFEAQHSQRNPQDAAGR 127 LILRB1 Q8NHL6MTPILTVLICLGLSLGPRTHVQAGHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTALWITRIPQELVKKGQFPIPSITWEHAGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPSPVVNSGGNVILQCDSQVAFDGFSLCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLGVSKKPSLSVQPGPIVAPEETLTLQCGSDAGYNRFVLYKDGERDFLQLAGAQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSEWSAPSDPLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQGWMQTFLLTKEGAADDPWRLRSTYQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKPYLLTHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH 128 LILRB2 Q8N423MTPIVTVLICLGLSLGPRTHVQTGTIPKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRLYREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRARWSELSDPLVLVMTGAYPKPTLSAQPSPVVTSGGRVTLQCESQVAFGGFILCKEGEEEHPQCLNSQPHARGSSRAIFSVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDLLELLVPGVSKKPSLSVQPGPVVAPGESLTLQCVSDVGYDRFVLYKEGERDLRQLPGRQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSECSAPSDPLDILITGQIRGTPFISVQPGPTVASGENVTLLCQSWRQFHTFLLTKAGAADAPLRLRSIHEYPKYQAEFPMSPVTSAHAGTYRCYGSLNSDPYLLSHPSEPLELVVSGPSMGSSPPPTGPISTPAGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVVLLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKDTQPEDGVEMDTRAAASEAPQDVTYAQLHSLTLRRKATEPPPS QEREPPAEPSIYATLAIH 129 LILRB2NP_00586 MTPIVTVLICLGLSLGPRTRVQTGTIPKPTLWAEPDSVITQGS 5.3PVTLSCQGSLEAQEYRLYREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRARWSELSDPLVLVMTGAYPKPTLSAQPSPVVTSGGRVTLQCESQVAFGGFILCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDLLELLVPGVSKKPSLSVQPGPVMAPGESLTLQCVSDVGYDRFVLYKEGERDLRQLPGRQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSECSAPSDPLDILITGQIRGTPFISVQPGPTVASGENVTLLCQSWRQFHTFLLTKAGAADAPLRLRSIHEYPKYQAEFPMSPVTSAHAGTYRCYGSLNSDPYLLSHPSEPLELVVSGPSMGSSPPPTGPISTPAGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVVLLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKDTQPEDGVEMDTRAAASEAPQDVTYAQLHSLTLRRKATEPPPS QEREPPAEPSIYATLAIH 130 LILRB3O75022 MTPALTALLCLGLSLGPRTRVQAGPFPKPTLWAEPGSVISWGSPVTIWCQGSQEAQEYRLHKEGSPEPLDRNNPLEPKNKARFSIPSMTEHHAGRYRCHYYSSAGWSEPSDPLEMVMTGAYSKPTLSALPSPVVASGGNMTLRCGSQKGYHHFVLMKEGEHQLPRTLDSQQLHSRGFQALFPVGPVTPSHRWRFTCYYYYTNTPWVWSHPSDPLEILPSGVSRKPSLLTLQGPVLAPGQSLTLQCGSDVGYNRFVLYKEGERDFLQRPGQQPQAGLSQANFTLGPVSPSNGGQYRCYGAHNLSSEWSAPSDPLNILMAGQIYDTVSLSAQPGPTVASGENVTLLCQSWWQFDTFLLTKEGAAHPPLRLRSMYGAHKYQAEFPMSPVTSAHAGTYRCYGSYSSNPHLLSHPSEPLELVVSGHSGGSSLPPTGPPSTPGLGRYLEVLIGVSVAFVLLLFLLLFLLLRRQRHSKHRTSDQRKTDFQRPAGAAETEPKDRGLLRRSSPAADVQEENLYAAVKDTQSEDRVELDSQSPHDEDPQAVTYAPVKHSSPRREMASPPSSLSGEFLDTKDRQVEEDRQMDTEAAASEASQDVTYAQLHSLTLRRKATEPPPS QEGEPPAEPSIYATLAIH 131 LILRB4Q8NHJ6 MIPTFTALLCLGLSLGPRTHMQAGPLPKPTLWAEPGSVISWGNSVTIWCQGTLEAREYRLDKEESPAPWDRQNPLEPKNKARFSIPSMTEDYAGRYRCYYRSPVGWSQPSDPLELVMTGAYSKPTLSALPSPLVTSGKSVTLLCQSRSPMDTFLLIKERAAHPLLHLRSEHGAQQHQAEFPMSPVTSVHGGTYRCFSSHGFSHYLLSHPSDPLELIVSGSLEDPRPSPTRSVSTAAGPEDQPLMPTGSVPHSGLRRHWEVLIGVLVVSILLLSLLLFLLLQHWRQGKHRTLAQRQADFQRPPGAAEPEPKDGGLQRRSSPAADVQGENFCAAVKNTQPEDGVEMDTRQSPHDEDPQAVTYAKVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSFTLRQKATEPPPSQEGASPAEPSVYATLAIH 132 LILRB5 O75023MTLTLSVLICLGLSVGPRTCVQAGTLPKPTLWAEPASVIARGKPVTLWCQGPLETEEYRLDKEGLPWARKRQNPLEPGAKAKFHIPSTVYDSAGRYRCYYETPAGWSEPSDPLELVATGFYAEPTLLALPSPVVASGGNVTLQCDTLDGLLTFVLVEEEQKLPRTLYSQKLPKGPSQALFPVGPVTPSCRWRFRCYYYYRKNPQVWSNPSDLLEILVPGVSRKPSLLIPQGSVVARGGSLTLQCRSDVGYDIFVLYKEGEHDLVQGSGQQPQAGLSQANFTLGPVSRSHGGQYRCYGAHNLSPRWSAPSDPLDILIAGLIPDIPALSVQPGPKVASGENVTLLCQSWHQIDTFFLTKEGAAHPPLCLKSKYQSYRHQAEFSMSPVTSAQGGTYRCYSAIRSYPYLLSSPSYPQELVVSGPSGDPSLSPTGSTPTPGPEDQPLTPTGLDPQSGLGRHLGVVTGVSVAFVLLLFLLLFLLLRHRHQSKHRTSAHFYRPAGAAGPEPKDQGLQKRASPVADIQEEILNAAVKDTQPKDGVEMDARAAASEAPQDVTYAQLHSLTLRREATEPPPSQEREP PAEPSIYAPLAIH 133 MacacaXP_01529 MTPILMVLICLGLSLGPRTHVQAGILPKPTLWAEPGSVISEGS fascicularis 7203PVTLRCQGSLQVQEYHLYREKNPASWVRQIRQELVKKGYFA LILRB2IGFITWEHTGQYRCQYYSHSWWSEPSDPLELVVTGAYSKPT (putative)LSALPSPVVASGGNVTLQCDSQVAFDSFTLCKEGEDEHPQRLNCQSHARGWSWAVFSVGPVSPSRRWSYRCYGYISSAPNVWSLPSDLLELLVPGVSKKPSLSVQPGPVVAPGDKLTLQCGSDAGYDRFALYKEGEGDFLQRPVRQPQAGLSQANFLLGPVSRSHGGQYRCSGAHNLSSEWSAPSDPLDILIAGQIRGRPFLSVQPGPKVVSGENVTLLCQSSWQFHAFLLTQAGAADAHLHLRSMYKYPKYQAEFPMSPVTSAHAGTYRCYGSRSSNPYLLSVPSDPLELVVSGPSGGPSSPTTGPTSTCAGPEDQPLTPTGSAPQSGLGRHLGVVTGVLVAFVLLLFLLLLLFLVLRYRRQGKRWTSAQRKADFQHPAGAVEPEPRDRGLQRRSSPAADTQEENLYAAVKDTQPEDGVELDSRAAASEDPQDVTYAQLQSLTLRREAT EPPPSQERAPPVESSIYATLTIH 134Macaca Q1I0P6_ GLSLGSRTRVQAGTLPKPTLWAEPDSVITQGSPVTLRCQGS mulatta MACMULQVQEYRLYRERKPASWVRRIRQELVKKGYFAIGFITWEHTG LILRBbQYHCQYYSHSWWPEPSDPLELVMTGAYSKPTLSALPSPMV (putative)ASGGNVTLQCDSQVAFDGFILCKEGEDEHPQRLNSHFHAYGWSRAVFSVGPVSPSRRWSYRCYGYDSRSPYVWSLPSDLLELLVPGVSKKPSLSVQPGPVVAPGDKLTLQCGSDAGYNRFALYKEGEGNFLQHPGRQRQAGLSQANFLLGPVSRSHGGQYRCYGAHNLSSEWSAPSDPLDILIAGQIRGRPSLLVQPGRTVASGENVTLLCQSSWQFHVFLLTQAGAADAHLHLRSMYKYPKYQAEFPMSPVTSAHAGTYRCYGSHSSDSYLLSVPSDPLELVVSGPSGGPSSPTTGPTSTCGPEDQPLTPTGSAPQSGLGRHLGVVTGVLVAFVLLLFLLLLLFLVLRHRRQGKRWTSAQRKADFQHPAGAVEPEPRDRGLQRRSSPAANTQEENLYAAMKDTQPEDGVELDSQAAASEDPQDVTYAQLQSLTLRRETTEPPPSQERA PPVESSIY

OTHER EMBODIMENTS

The disclosure may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting of the disclosure. Scope of the disclosure is thusindicated by the appended claims rather than by the foregoingdescription, and all changes that come within the meaning and range ofequivalency of the claims are therefore intended to be embraced herein.All references cited herein are incorporated herein by reference.

In the event of an inconsistency between a sequence in the sequencelisting and a sequence in the specification, the sequence in thespecification should be considered as controlling.

1-16. (canceled)
 17. An antibody that specifically binds to humanLILRB2, wherein the antibody comprises the following six complementaritydetermining regions (CDRs): (a) a CDR-H1 comprising the amino acidsequence of SEQ ID NO: 15; (b) a CDR-H2 comprising the amino acidsequence of SEQ ID NO: 16; (c) a CDR-H3 comprising the amino acidsequence of SEQ ID NO: 17; (d) a CDR-L1 comprising the amino acidsequence of SEQ ID NO: 18; (e) a CDR-L2 comprising the amino acidsequence of SEQ ID NO: 19; and (f) a CDR-L3 comprising the amino acidsequence of SEQ ID NO:
 20. 18. The antibody of claim 17, wherein theantibody comprises a variable heavy chain (VH) region comprising anamino acid sequence that is at least 90% identical to the amino acidsequence of SEQ ID NO: 13 and a variable light chain (VL) regioncomprising an amino acid sequence that is at least 90% identical to theamino acid sequence of SEQ ID NO: 14, wherein the VH region comprisesthree CDRs comprising the amino acid sequences of SEQ ID NOs: 15-17, andthe VL region comprises three CDRs comprising the amino acid sequencesof SEQ ID NOs: 18-20.
 19. The antibody of claim 17, wherein the antibodycomprises a VH region comprising the amino acid sequence of SEQ ID NO:13 and a VL region comprising the amino acid sequence of SEQ ID NO: 14.20. The antibody of claim 17, wherein the antibody comprises a heavychain comprising the amino acid sequence of SEQ ID NO: 11 and a lightchain comprising the amino acid sequence of SEQ ID NO:
 12. 21. Theantibody of claim 17, wherein the antibody comprises a VH regioncomprising an amino acid sequence that is at least 90% identical to theamino acid sequence of SEQ ID NO: 53 and a VL region comprising an aminoacid sequence that is at least 90% identical to the amino acid sequenceof SEQ ID NO: 54, wherein the VH region comprises three CDRs comprisingthe amino acid sequences of SEQ ID NOs: 15-17, and the VL regioncomprises three CDRs comprising the amino acid sequences of SEQ ID NOs:18-20.
 22. The antibody of claim 17, wherein the antibody comprises a VHregion comprising the amino acid sequence of SEQ ID NO: 53 and avariable light chain VL region comprising the amino acid sequence of SEQID NO:
 54. 23. The antibody of claim 17, wherein the antibody comprisesa heavy chain comprising the amino acid sequence of SEQ ID NO: 51 and alight chain comprising the amino acid sequence of SEQ ID NO:
 52. 24. Theantibody of claim 17, wherein the antibody comprises a VH regioncomprising an amino acid sequence that is at least 90% identical to theamino acid sequence of SEQ ID NO: 63 and a VL region comprising an aminoacid sequence that is at least 90% identical to the amino acid sequenceof SEQ ID NO: 64, wherein the VH region comprises three CDRs comprisingthe amino acid sequences of SEQ ID NOs: 15-17, and the VL regioncomprises three CDRs comprising the amino acid sequences of SEQ ID NOs:18-20.
 25. The antibody of claim 17, wherein the antibody comprises a VHregion comprising the amino acid sequence of SEQ ID NO: 63 and a VLregion comprising the amino acid sequence of SEQ ID NO:
 64. 26. Theantibody of claim 17, wherein the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 61 and a light chaincomprising the amino acid sequence of SEQ ID NO:
 62. 27. The antibody ofclaim 17, wherein the antibody comprises a VH region comprising an aminoacid sequence that is at least 90% identical to the amino acid sequenceof SEQ ID NO: 73 and a VL region comprising an amino acid sequence thatis at least 90% identical to the amino acid sequence of SEQ ID NO: 74,wherein the VH region comprises three CDRs comprising the amino acidsequences of SEQ ID NOs: 15-17, and the VL region comprises three CDRscomprising the amino acid sequences of SEQ ID NOs: 18-20.
 28. Theantibody of claim 17, wherein the antibody comprises a VH regioncomprising the amino acid sequence of SEQ ID NO: 73 and a VL regioncomprising the amino acid sequence of SEQ ID NO:
 74. 29. The antibody ofclaim 17, wherein the antibody comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 71 and a light chain comprising theamino acid sequence of SEQ ID NO:
 72. 30. The antibody of claim 17,wherein the antibody comprises a VH region comprising an amino acidsequence that is at least 90% identical to the amino acid sequence ofSEQ ID NO: 83 and a VL region comprising an amino acid sequence that isat least 90% identical to the amino acid sequence of SEQ ID NO: 84,wherein the VH region comprises three CDRs comprising the amino acidsequences of SEQ ID NOs: 15-17, and the VL region comprises three CDRscomprising the amino acid sequences of SEQ ID NOs: 18-20.
 31. Theantibody of claim 17, wherein the antibody comprises a VH regioncomprising the amino acid sequence of SEQ ID NO: 83 and a VL regioncomprising the amino acid sequence of SEQ ID NO:
 84. 32. The antibody ofclaim 17, wherein the antibody comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 81 and a light chain comprising theamino acid sequence of SEQ ID NO:
 82. 33-49. (canceled)
 50. The antibodyof claim 17, wherein the heavy chain additionally comprises a C-terminallysine. 51-66. (canceled)
 67. The antibody of claim 17, wherein theantibody binds to LILRB2 with a dissociation constant (K_(D)) of lessthan 3.0 nM.
 68. The antibody of claim 67, wherein the antibody binds toLILRB2 with a K_(D) of less than 1.5 nM.
 69. The antibody of claim 68,wherein the antibody binds to LILRB2 with a K_(D) of less than 1.0 nM.70. The antibody of claim 69, wherein the antibody binds to LILRB2 witha dissociation constant (K_(D)) less than 750 μM.
 71. The antibody ofclaim 17, wherein the antibody is a monoclonal antibody.
 72. Theantibody of claim 17, wherein the antibody is a chimeric antibody, ahumanized antibody, a CDR-grafted antibody, or a human antibody.
 73. Theantibody of claim 17, wherein the antibody comprises an Fc regionselected from the group consisting of a native Fc region, a variant Fcregion, and a functional Fc region.
 74. The antibody of claim 17,wherein the antibody is a conjugate antibody or is detectably labeled.75. The antibody of claim 17, wherein the antibody is an IgG1 antibody,an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.
 76. Theantibody of claim 75, which is an IgG4 antibody. 77-78. (canceled)
 79. Apharmaceutical composition comprising the antibody of claim
 17. 80-96.(canceled)
 97. An antibody that specifically binds to human LILRB2,wherein the antibody comprises (a) a VH region comprising an amino acidsequence that is at least 98% identical to the amino acid sequence ofSEQ ID NO: 13 and a VL region comprising an amino acid sequence that isat least 98% identical to the amino acid sequence of SEQ ID NO: 14; (b)a VH region comprising an amino acid sequence that is at least 98%identical to the amino acid sequence of SEQ ID NO: 63 and a VL regioncomprising an amino acid sequence that is at least 98% identical to theamino acid sequence of SEQ ID NO: 64; (c) a VH region comprising anamino acid sequence that is at least 98% identical to the amino acidsequence of SEQ ID NO: 73 and a VL region comprising an amino acidsequence that is at least 98% identical to the amino acid sequence ofSEQ ID NO: 74; or (d) a VH region comprising an amino acid sequence thatis at least 98% identical to the amino acid sequence of SEQ ID NO: 83and a VL region comprising an amino acid sequence that is at least 98%identical to the amino acid sequence of SEQ ID NO:
 84. 98. An antibodythat specifically binds to human LILRB2, wherein the antibody comprisesa VH region comprising an amino acid sequence that is at least 98%identical to the amino acid sequence of SEQ ID NO: 53 and a VL regioncomprising an amino acid sequence that is at least 98% identical to theamino acid sequence of SEQ ID NO: 54.